Systems, articles, and methods for flowing particles

ABSTRACT

Systems and methods for flowing particles, such as biological entities, in a fluidic channel(s) are generally provided. In some cases, the systems described herein are designed such that a single particle may be isolated from a plurality of particles and flowed into a fluidic channel (e.g., a microfluidic channel) and/or collected e.g., on fluidically isolated surfaces. For example, the single particle may be present in a plurality of particles of relatively high density and the single particle is flowed into a fluidic channel, such that it is separated from the plurality of particles. The particles may be spaced within a fluidic channel so that individual particles may be measured/observed over time. In certain embodiments, the particle may be a biological entity. Such article and methods may be useful, for example, for isolating single cells into individual wells of multi-well cell culture dishes (e.g., for single-cell analysis).

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/940,001, filed Mar. 29, 2018, and entitled “Systems, Articles, andMethods for Flowing Particles,” which claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application Ser. No. 62/480,148, filedMar. 31, 2017, and entitled “Systems and Methods for Flowing Particles,”to U.S. Provisional Patent Application Ser. No. 62/480,170, filed Mar.31, 2017, and entitled “Methods and Articles for Isolating SingleParticles,” and to U.S. Provisional Patent Application Ser. No.62/480,185, filed Mar. 31, 2017, and entitled “Devices and Methods forDirecting Flow of Particles,” each of which is incorporated herein byreference in its entirety for all purposes.

GOVERNMENT SUPPORT

This invention was made with Government support under Grant No. R01CA170592 awarded by the National Institutes of Health (NIH). TheGovernment has certain rights in the invention.

TECHNICAL FIELD

The present invention generally relates to methods, systems, articles,and devices for flowing particles, such as biological entities, in afluidic channel. In some cases, the invention relates to directing flowof particles in a fluidic channel and/or isolating particles onfluidically isolated surfaces.

SUMMARY

The present invention generally relates to methods, systems, articles,and devices methods for flowing particles, such as biological entities,in a fluidic channel, directing flow of particles in a fluidic channel,and isolating particles on fluidically isolated surfaces. In one aspect,methods are provided. In some embodiments, the method comprises flowinga plurality of particles in a first fluidic channel such that a singleparticle enters a second fluidic channel, wherein the second fluidicchannel intersects and is in fluidic communication with the firstfluidic channel, detecting, with a detector, the presence of the singleparticle in the second fluidic channel, and upon detecting the presenceof the single particle in the second fluidic channel, flowing at least aportion of the remaining plurality of particles through the firstfluidic channel, while maintaining the single particle in the secondfluidic channel and introducing no additional particles into the secondfluidic channel.

In some embodiments, the method comprises introducing a single particleinto a second fluidic channel from a first fluidic channel containing aplurality of particles, the second fluidic channel in fluidiccommunication with the first fluidic channel, detecting the singleparticles in the second fluidic channel, and responsive to detecting thesingle particle, retaining the single particle essentially at a constantflow rate in the second fluidic channel while flowing additionalparticles through the first fluidic channel.

In some embodiments, the method comprises introducing a plurality ofparticles into a first fluidic channel, flowing the plurality ofparticles in the first fluidic channel such that at least a portion ofthe particles enter a second fluidic channel, wherein the second fluidicchannel intersects and is in fluidic communication with the firstfluidic channel, wherein each particle enters the second fluidic channelfrom the first fluidic channel at a frequency within a range of fromless than or equal to 1 particle per 10 seconds to greater than or equalto 1 particle per 120 seconds, and flowing, between the entry of eachparticle into the second fluidic channel from the first fluidic channel,a fluid in the first fluidic channel.

In some embodiments, the method comprises introducing a fluid comprisingplurality of particles to a first fluidic channel and flowing the fluidin the first fluidic channel such that at least a portion of theparticles enter a second fluidic channel, wherein the second fluidicchannel intersects and is in fluidic communication with the firstfluidic channel, wherein each particle enters the second fluidic channelfrom the first fluidic channel at a frequency of less than or equal to 1particle per 10 particles are present in the fluid at a density of atleast 100 particles per mL.

In some embodiments, the method comprises introducing, from a firstfluidic channel containing a disordered arrangement of particles, into asecond fluidic channel, a series of individual particles positioned inthe second fluidic channel, separated from each other by a spacing withan average distance of from 20 microns to 500 mm, wherein 90% of thespacings differ by no more than 10% from the average distance, at a rateof at least 1 particles per 10 seconds.

In some embodiments, the method comprises flowing, through a fluidicchannel associated with a plurality of fluidically isolated surfaces, aplurality of particles and collecting the plurality of particles, suchthat each particle is associated with a single fluidically isolatedsurface.

In some embodiments, the method comprises flowing, through a fluidicchannel, a plurality of particles such that each particle is spaced atleast 1 mm apart along the longitudinal axis of the fluidic channel andcollecting each particle on a fluidically isolated surface.

In another aspect, methods for collecting particles are provided. Insome embodiments, the method comprises flowing, through a second fluidicchannel, a plurality of particles and flowing, through a first fluidicchannel in fluidic communication with the second fluidic channel, atleast a portion of the plurality of particles, wherein the secondfluidic channel comprises an exit positioned in the first fluidicchannel and wherein a frequency of particles exiting the second fluidicchannel into the first fluidic channel is less than or equal to 1particle per 10 seconds and greater than or equal to 1 particle per 120seconds.

In another aspect, systems are provided. In some embodiments, the systemcomprises a first fluidic channel, a second fluidic channel intersectingand in fluidic communication with the first fluidic channel, at leastone pressure source associated with the first fluidic channel, and adetector associated with the second fluidic channel, wherein the systemis configured such that, upon detection by the detector of the presenceof a single particle in the second fluidic channel, at least oneproperty of one or more of the at least one pressure source is changed.

In another aspect, articles are provided. In some embodiments, thearticle comprises a plurality of fluidically isolated surfaces and asingle particle associated with each fluidically isolated surface.

In one aspect, fluidic devices are provided. In some embodiments, thefluidic device comprises a suspended microchannel resonator, a secondfluidic channel in fluidic communication with the suspended microchannelresonator, and a first fluidic channel in fluidic communication with thesecond fluidic channel, wherein a longitudinal axis of the secondfluidic channel is orthogonal to a longitudinal axis of the firstfluidic channel and the second fluidic channel comprises an exitpositioned at or near the center of the first fluidic channel.

In certain embodiments, the plurality of particles are a plurality ofbiological entities. In certain embodiments, the plurality of biologicalentities comprise virions, bacteria, protein complexes, exosomes, cells,or fungi.

Other advantages and novel features of the present invention will becomeapparent from the following detailed description of various non-limitingembodiments of the invention when considered in conjunction with theaccompanying figures. In cases where the present specification and adocument Incorporated by reference include conflicting and/orinconsistent disclosure, the present specification shall control.

BACKGROUND

Single-cell analysis is a powerful approach in advancing understandingof health and disease. For example, in cancer biology, tumors consist ofgenetically heterogeneous cell populations that are difficult to addresswith traditional bulk tumor measurements. Recent technologicalprogresses reveal growing applications of single-cell analysis in cancertranslational medicine such as early detection, diagnosis, treatmentmonitoring and selection. However, there remain significant challengeswith single-cell isolation with respect to yield, quality, throughput,and cost.

Accordingly, improved devices and methods are needed.

BRIEF DESCRIPTION OF THE DRAWINGS

Non-limiting embodiments of the present invention will be described byway of example with reference to the accompanying figures, which areschematic and are not intended to be drawn to scale. In the figures,each identical or nearly identical component illustrated is typicallyrepresented by a single numeral. For purposes of clarity, not everycomponent is labeled in every figure, nor is every component of eachembodiment of the invention shown where illustration is not necessary toallow those of ordinary skill in the art to understand the invention. Inthe figures:

FIG. 1 is a schematic illustration of a system for flowing a particle,according to one set of embodiments;

FIG. 2A is a cross-sectional top-down view schematic illustration of adevice for directing the flow of a particle, according to one set ofembodiments;

FIG. 2B is a cross-sectional side view schematic illustration of adevice for directing the flow of particle, according to one set ofembodiments;

FIG. 2C is a cross-sectional schematic illustration of a device fordirecting the flow of a particle, according to one set of embodiments;

FIG. 3A is a schematic illustration of a system for flowing a particle,according to one set of embodiments;

FIG. 3B is a schematic illustration of a system for flowing a particle,according to one set of embodiments;

FIG. 3C is a schematic illustration of a system for flowing a particle,according to one set of embodiments;

FIG. 3D is a schematic illustration of a system for flowing a particle,according to one set of embodiments;

FIG. 3E is a schematic illustration of a system for flowing a particle,according to one set of embodiments;

FIG. 4 is a schematic illustration of a system for determining aproperty of a particle, according to one set of embodiments;

FIG. 5 is a top-down view schematic illustration of an articlecomprising isolated particles in a plurality of fluidically isolatedsurfaces, according to one set of embodiments;

FIG. 6 is a perspective view schematic illustration of an articlecomprising isolated particles in a plurality of fluidically isolatedsurfaces, according to one set of embodiments;

FIG. 7 is a perspective view schematic illustration of a system forcollecting particles in fluidically isolated surfaces, according to oneset of embodiments;

FIG. 8A is a schematic illustration of an exemplary system fordetermining a property of a particle, according to one set ofembodiments;

FIG. 8B is a schematic illustration of the relative flow resistance influidic channels of the system, according to one set of embodiments;

FIG. 8C is a fluid flow simulation of a system under a particle‘loading’ regime (such as that illustrated in FIG. 8A), according to oneset of embodiments;

FIG. 8D is a fluid flow simulation of a system under a particle‘flushing’ regime (such as that illustrated in FIG. 8B), according toone set of embodiments; and

FIG. 9 are plots of resonance frequency of an SMR versus time forpassive loading and active loading of particles, according to one set ofembodiments.

FIG. 10A is a micrograph of an exemplary biological entity isolated on afluidically isolated surface and monitored for growth over 8 days on theisolated surface, according to one set of embodiments; and

FIG. 10B is a micrograph of an exemplary biological entity isolated on afluidically isolated surface and monitored for growth over 8 days on theisolated surface, according to one set of embodiments; and

FIG. 11 is a fluid flow simulation of a comparative device comprisingtwo intersecting channels, according to one set of embodiments; and

FIG. 12 is a schematic illustration of an exemplary device for directingthe flow of a particle, according to one set of embodiments.

DETAILED DESCRIPTION

Systems and methods for flowing particles, such as biological entities,in a fluidic channel(s) are generally provided. In some cases, thesystems described herein are designed such that a single particle may beisolated from a plurality of particles and flowed into a fluidic channel(e.g., a microfluidic channel). For example, the single particle may bepresent in a plurality of particles of relatively high density and thesingle particle is flowed into a fluidic channel, such that it isseparated from the plurality of particles. In some cases, more than oneparticle may be flowed into a fluidic channel such that each particleenters the fluidic channel at a relatively low frequency (e.g., of lessthan 1 particle per 10 seconds). The particles may be spaced within afluidic channel so that individual particles may be measured/observedover time. In certain embodiments, the particle may be a biologicalentity.

In some embodiments, devices and methods for directing the flow ofparticles, such as biological entities, in a fluidic channel(s) areprovided. In some cases, the devices described herein are designed suchthat a single particle may be collected from a plurality of particlesand flowed into a fluidic channel (e.g., a microfluidic channel). Forexample, the single particle may be present in a fluidic channelcomprising a plurality of particles and the single particle is flowedinto an orthogonal fluidic channel, such that the single particle iscollected (e., via an outlet of the orthogonal fluidic channel). In somecases, more than one particle may be flowed into a second fluidicchannel from a first fluidic channel such that each particle enters thesecond fluidic channel at a relatively low frequency (e.g., of less than1 particle per 10 seconds). One or more physical properties of theparticles may be measured/observed over time before collecting theparticle(s). In certain embodiments, the particle may be a biologicalentity.

Certain embodiments are related to methods and articles for isolatingparticles such as biological entities on, for example, fluidicallyisolated surfaces. In some cases, the methods and articles are designedsuch that a single particle isolated from a plurality of particles maybe associated with a single fluidically isolated surface amongst aplurality of fluidically isolated surfaces. Such article and methods maybe useful, for example, for isolating single cells into individual wellsof multi-well cell culture dishes (e.g., for single-cell analysis). Incertain embodiments, a plurality of particle are flowed along a channelat a particular spacing, such that a single particle may be introducedonto a fluidically isolated surface.

The term ‘fluidically isolated surface’ as used herein refers a surfacewhich is not in liquid communication with another surface. A surfacethat is fluidically isolated with respect to another surface refers to asurface of the same type (e.g., the bottom surface of a well, the bottomsurface of a dish, the sidewall of a conical tube). As used herein, a“fluid” is given its ordinary meaning, i.e., a liquid or a gas. A fluidcannot maintain a defined shape and will flow during an observable timeframe to fill the container in which it is put. Thus, the fluid may haveany suitable viscosity that permits flow. However, one of ordinary skillin the art would understand, based upon the teachings of thisspecification, that when two or more surfaces are said to be‘fluidically isolated’, that this refers to two or more surfaces (of thesame type) that are specifically not in liquid communication. Those ofordinary skill in the art would also understand that a liquid need notbe present for the two or more surfaces to be ‘fluidically isolated’.For example, in some embodiments, the introduction of a liquid to one ofthe two or more fluidically isolated surfaces, would not result in theliquid being introduced to any of the remaining surfaces. The two ormore surfaces may, in some cases, be ‘fluidically isolated’ and ingaseous communication (e.g., two or more surfaces exposed to the samesurrounding environment). In some embodiments, the two or more surfacesmay be physically connected (e.g., two or more wells of a multi-wellcell culture plate), however, each surface is fluidically isolated fromone another. In some embodiments, the two or more surfaces may not bephysically connected (e.g., surfaces present on two or more conicaltubes, two or more channels, two or more dishes (e.g., petri dishes)).In certain embodiments, single particles from a plurality of particlesmay each be associated with each fluidically isolated surface amongst aplurality of fluidically isolated surfaces.

In an exemplary embodiment, a first fluidically isolated surfacecomprises the bottom surface of a first well (e.g., of a multi-well cellculture plate) and a second fluidically isolated surface comprises thebottom surface of a second well. One of ordinary skill in the art wouldunderstand that while each well may comprise, for example, a sidewall(i.e. a surface in physical and fluidic communication with the bottomsurface of the well), that fluidically isolated surfaces refers tofluidically isolated surfaces of the same type (e.g., the bottom surfaceof each well).

In another exemplary embodiment, a first fluidically isolated surfacecomprises a first hydrophilic region of a substrate and a secondfluidically isolated surface comprises a second hydrophilic region ofthe substrate, such that the first hydrophilic region and the secondhydrophilic region are not in liquid communication.

Advantageously, the systems, devices, and methods described herein maypermit the measurement and/or observation of a single particle (e.g., abiological entity such as a cell or bacteria) over relatively longperiods of time (e.g., greater than 10 minutes). For example, the growthof a cell may be monitored using an array of suspended microchannelresonators and the use of the systems and methods described hereinpermit the measurement of a single cell in a single suspendedmicrochannel resonator at a particular moment in time. In someembodiments, the spacing (or frequency) of particles within the fluidicchannel may be controlled. Advantageously, the particles may beseparated from a relatively concentrated source of particles withoutsubsequent and/or significant dilution of the source and/or without theapplication of relatively high shear forces being applied to theparticles. For example, a relatively high density plurality of particles(e.g., biological entities) may be introduced to a fluidic channel and asingle particle from the plurality of particles may be flowed into anintersecting fluidic channel and separated from the plurality ofparticles without diluting the plurality of particles. In some suchcases, multiple particles may be flowed into the intersecting fluidicchannel from the plurality of particles such that each particle in theintersecting fluidic channel are spaced apart with a relatively uniformand large average spacing (e.g., at least 1 mm apart) and/or enter thefluidic channel at a relatively low average frequency (e.g., less thanor equal to 1 particle per 10 seconds). Such systems and methods may beparticularly useful for measuring the physical properties (e.g., mass,size, density, or change of mass, size, and/or density over time) ofindividual cells (e.g., bacteria, yeast, liquid tumor cells, solid tumorcells suspended in fluid, immune cells). Such systems and methods mayalso be useful for measuring physical properties of populations of cellsthat are physically attached to each other such as neuro-spheres ofglioblastoma multiforme or masses of tumor cells that are isolated froma tissue biopsy or cell culture.

Advantageously, the devices and methods described herein may permit thecollection of individual particles (e.g., biological entities) at adesired frequency (e.g., such that the individual particles may becollected and/or sorted). In some embodiments, a plurality of particlesmay be collected such that two or more particles do not aggregate duringcollection (e.g., a single particle exits an outlet of the device at aparticular frequency). In some cases, the growth of a cell may bemonitored using an array of suspended microchannel resonators and theuse of the devices and methods described herein permit the collection ofa single cell after determination of one or more physical properties ofthe cell.

In some embodiments, the particle is a biological entity. Non-limitingexamples of biological entities include virions, bacteria, proteincomplexes, exosomes, cells, or fungi (e.g., yeast). In some embodiments,the biological entity is obtained from a subject. A “subject” refers toany animal such as a mammal (e.g., a human). Non-limiting examples ofsubjects include a human, a non-human primate, a cow, a horse, a pig, asheep, a goat, a dog, a cat or a rodent such as a mouse, a rat, ahamster, a bird, a fish, or a guinea pig. In an exemplary embodiment,the biological entity is a human cell. In some embodiments, the systemsand methods described herein are useful for separating biologicalentities into a fluidic channel from a plurality of biological entitiesobtained from a subject for example, determining one or more physicalproperties of the biological entity (e.g., growth behavior), sorting,and/or diagnostic purposes.

In some embodiments, the plurality of particles (e.g., a plurality ofbiological entities) are provided (e.g., suspended) in a fluid. In someembodiments, the plurality of particles are in disordered arrangement inthe fluid. As used herein, a “fluid” is given its ordinary meaning,i.e., a liquid or a gas. A fluid cannot maintain a defined shape andwill flow during an observable time frame to fill the container in whichit is put. Thus, the fluid may have any suitable viscosity that permitsflow. In a particular set of embodiments, the fluid is a liquid. In someembodiments, the fluid comprises water, a reagent, a solvent, a buffer,a cell-growth medium, or combinations thereof. In certain embodiments,the particles are relatively soluble in the fluid. In some embodiments,the fluid does not comprise a colloid (e.g., such as an emulsion). Forexample, in some embodiments, the particle (e.g., biological entity) isnot disposed (and/or encapsulated) by a first fluid that is immisciblewith, and surrounded by, a second fluid different than the first fluid.However, one of ordinary skill in the art, based upon the teachings ofthis specification, would understand that the systems and methodsdescribed herein may be used for separating a single colloid into achannel from a plurality of colloids. One of ordinary skill in the art,based upon the teachings of this specification, would also understandthat the devices and methods described herein may be used for collectinga single colloid in a channel from a plurality of colloids.

As illustrated in FIG. 1, in some embodiments, system 100 comprisesfirst fluidic channel 110 (e.g., a primary fluidic channel) and secondfluidic channel 120 (e.g., an intersection fluidic channel) intersectingfirst fluidic channel 110 at intersection 130. In some such embodiments,second fluidic channel 120 is downstream of, and terminates at,intersection 130. Those of ordinary skill in the art would understandthat while FIG. 1 illustrates second fluidic channel 120 as orthogonalto first fluidic channel 110, such illustration is intended to benon-limiting and that any suitable non-zero angle between first fluidicchannel 110 and second fluidic channel 120 may also be possible (e.g.,an angle between first fluidic channel 110 and second fluidic channel120 of greater than or equal to 15 degrees and less than or equal to 90degrees, greater than or equal to 15 degrees and less than or equal to45 degrees, greater than or equal to 30 degrees and less than or equalto 60 degrees, greater than or equal to 45 degrees and less than orequal to 75 degrees, greater than or equal to 60 degrees and less thanor equal to 90 degrees, or greater than or equal to 75 degrees and lessthan or equal to 90 degrees).

As illustrated in FIG. 2A, in some embodiments, device 100 comprises afirst fluidic channel 110 (e.g., a collection channel) and a secondfluidic channel 120 (e.g., an outlet channel) comprising an exit 130(e.g., an outlet) positioned at or near the center (e.g., as indicatedby centerline 115) of at least one cross-sectional dimension of firstfluidic channel 110. In some embodiments, a longitudinal axis of secondfluidic channel 120 is orthogonal to a longitudinal axis of firstfluidic channel 110. In certain embodiments, an exit 130 of secondfluidic channel 120 is disposed within first fluidic channel 110 (e.g.,such that first fluidic channel 110 and second fluidic channel 120 arein fluidic communication). Those of ordinary skill in the art wouldunderstand that while FIG. 2A illustrates second fluidic channel 120 asorthogonal to first fluidic channel 110, such illustration is intendedto be non-limiting and that any suitable non-zero angle between firstfluidic channel 110 and second fluidic channel 120 may also be possible(e.g., an angle between first fluidic channel 110 and second fluidicchannel 120 of greater than or equal to 15 degrees and less than orequal to 90 degrees, greater than or equal to 15 degrees and less thanor equal to 45 degrees, greater than or equal to 30 degrees and lessthan or equal to 60 degrees, greater than or equal to 45 degrees andless than or equal to 75 degrees, greater than or equal to 60 degreesand less than or equal to 90 degrees, or greater than or equal to 75degrees and less than or equal to 90 degrees).

In some embodiments, at least a portion of second fluidic channel mayhave a longitudinal axis that is substantially parallel to alongitudinal axis of the first fluidic channel. In some cases, theportion of the second fluidic channel that has a longitudinal axis thatis substantially parallel to a longitudinal axis of the first fluidicchannel may be disposed within the first fluidic channel.

In certain embodiments, the exit of the second fluidic channel isoriented orthogonal to the direction of flow of a fluid in the firstfluidic channel. For example, as illustrated in FIG. 2B, exit 130 isorthogonal to the direction of flow (e.g., the direction of fluid of afluid in first channel 110 as indicated by arrow 160 in FIG. 2A) of afluid in first channel 110. In an exemplary embodiment, a particle(e.g., exemplary particle 140) follows fluidic flow path out of the exitof the second fluidic channel into the first fluidic channel that is (orat least a portion of is) orthogonal to the fluidic flow path of a fluidin the first fluidic channel. For example, in an exemplary embodiment,exemplary particle 140 follows fluidic flow path 135 out of exit 130 ofsecond fluidic channel 120. In some embodiments, after exiting exit 130of second fluidic channel 120 into first fluidic channel 110, exemplaryparticle 140 flows in a direction orthogonal to the direction of fluidicflow in second fluidic channel 120 (e.g., exemplary particle 140 flowsin direction indicated by arrow 160 in FIG. 2A). Advantageously, incertain embodiments, the flow of one or more particles in first fluidicchannel 110 (after exiting exit 130) is directed (e.g., focused) at ornear the center (e.g., longitudinal axis 115 of first fluidic channel110) of the first fluidic channel.

Those of ordinary skill in the art would understand that while FIG. 2Aillustrates exit 130 as orthogonal to the direction of flow (e.g., arrow160) of first fluidic channel 110, such illustration is intended to benon-limiting and that any suitable non-zero angle between exit 130 andfirst fluidic channel 110 may also be possible (e.g., an angle betweenfirst fluidic channel 110 and exit 130 of greater than or equal to 15degrees and less than or equal to 90 degrees, greater than or equal to15 degrees and less than or equal to 45 degrees, greater than or equalto 30 degrees and less than or equal to 60 degrees, greater than orequal to 45 degrees and less than or equal to 75 degrees, greater thanor equal to 60 degrees and less than or equal to 90 degrees, or greaterthan or equal to 75 degrees and less than or equal to 90 degrees). Insome embodiments, as illustrated in FIG. 2A, first fluidic channel 110comprises outlet 170. In certain embodiments, a particle may becollected from outlet 170 (e.g., into a vessel such as a conical tube,petri-dish, or the like). In some such embodiments, one or moreparticles may be flowed in second fluidic channel 120 and exit secondfluidic channel 120 via exit 130 into first fluidic channel 110, suchthat it may be collected at outlet 170. In some embodiments, at leastone pressure source (e.g., first pressure source 190, second pressuresource 195) is associated with and/or in fluidic communication with thefirst fluidic channel. For example, as illustrated in FIG. 1, in someembodiments, first pressure source 190 is in fluidic communication withfirst fluidic channel 110. In certain embodiments, second pressuresource 195 is in fluidic communication with second fluidic channel 110.In some embodiments, first pressure source 190 and/or second pressuresource 195 are upstream of second fluidic channel 120 (e.g., such that,upon application of pressure to a fluid in fluidic channel 110, at leasta portion of the fluid enters fluidic channel 120). Each pressure sourcemay comprise any suitable means for providing pressure to a fluiddisposed within fluidic channel 110. For example, in some embodiments,each pressure source may be a pump such as a syringe pump, a suctionpump, a vacuum pump, a gas source, or any other suitable pressuresource, (e.g., which may act like a source or a sink). In someembodiments, each pressure source may not be in direct fluidiccommunication with the first fluidic channel. That is to say, in certainembodiments, one or more intervening fluidic channel(s) or fluidicregion(s) (e.g., fluidic reservoirs) of the device may be presentbetween the pressure source and the first fluidic channel.

Referring to FIG. 2A, in some embodiments, pressure source 192 is influidic communication with first fluidic channel 110. In someembodiments, pressure source 192 is upstream of second fluidic channel120 and applied to a fluid in first fluidic channel 110 (e.g., suchthat, upon application of pressure to a fluid in first fluidic channel110, a particle exiting exit 130 of second fluidic channel 120 iscaptured by the flow of the fluid). The pressure source may comprise anysuitable means for providing pressure to a fluid disposed within fluidicchannel 110. For example, in some embodiments, the pressure source maybe a pump such as a syringe pump, a suction pump, a vacuum pump, a gassource, or any other suitable pressure source, (e.g., which may act likea source or a sink). In some embodiments, the pressure source may not bein direct fluidic communication with the first fluidic channel. That isto say, in certain embodiments, one or more intervening fluidicchannel(s) or fluidic region(s) (e.g., fluidic reservoirs) of the devicemay be present between the pressure source and the first fluidicchannel.

In certain embodiments, each channel (e.g., first fluidic channel 110and/or second fluidic channel 120) of the system has a particularaverage cross-sectional dimension. The “cross-sectional dimension”(e.g., a width, a height, a radius) of the channel is measuredperpendicular to the direction of fluid flow. In some embodiments, theaverage cross-sectional dimension of one or more fluidic channels (e.g.,the first fluidic channel, the second fluidic channel) is less than orequal to 2 mm, less than or equal to 1 mm, less than or equal to 800microns, less than or equal to 600 microns, less than or equal to 500microns, less than or equal to 400 microns, less than or equal to 300microns, less than or equal to 200 microns, less than or equal to 100microns, less than or equal to 50 microns, less than or equal to 25microns, less than or equal to 20 microns, less than or equal to 15microns, or less than or equal to 10 microns. In certain embodiments,the average cross-sectional dimension of the channel is greater than orequal to 5 microns, greater than or equal to 10 microns, greater than orequal to 15 microns, greater than or equal to 20 microns, greater thanor equal to 25 microns, greater than or equal to 50 microns, greaterthan or equal to 100 microns, greater than or equal to 200 microns,greater than or equal to 300 microns, greater than or equal to 400microns, greater than or equal to 500 microns, greater than or equal to600 microns, greater than or equal to 800 microns, or greater than orequal to 1 mm. Combinations of the above-referenced ranges are alsopossible (e.g., greater than or equal to 5 microns and less than orequal to 2 mm, greater than or equal to 50 microns and less than orequal to 2 mm). Other ranges are also possible. In some embodiments, oneor more channels may be a microfluidic channel. “Microfluidic channels”generally refer to channels having an average cross-sectional dimensionof less than 1 mm.

In some embodiments, a ratio of the average cross-sectional dimension ofthe first fluidic channel and the average cross-sectional dimension ofthe second fluidic channel intersecting the first fluidic channel may bedesigned such that the resistance to flow into the second fluidicchannel may be controlled. For example, the ratio of the averagecross-sectional dimension of the first fluidic channel to the averagecross-sectional dimension of the second fluidic channel may be designedsuch that the system has a lower resistance to flow in the first fluidicchannel than the resistance to flow of in the second fluidic channelintersecting the first fluidic channel for a given pressure drop. Insome embodiments, the ratio of the average cross-sectional dimension ofthe first fluidic channel to the average cross-sectional dimension ofthe second fluidic channel is at least 1, at least 1.25, at least 1.5,at least 1.75, at least 2, at least 2.5, at least 3, at least 3.5, atleast 4, at least 5, at least 6, at least 7, at least 8, or at least 9.In certain embodiments, the ratio of the average cross-sectionaldimension of the first fluidic channel to the average cross-sectionaldimension of the second fluidic channel is less than or equal to 10,less than or equal to 9, less than or equal to 8, less than or equal to7, less than or equal to 6, less than or equal to 5, less than or equalto 4, less than or equal to 3.5, less than or equal to 3, less than orequal to 2.5, less than or equal to 2, less than or equal to 1.75, lessthan or equal to 1.5, or less than or equal to 1.25. Combinations of theabove-referenced ranges are also possible (e.g., at least 1 and lessthan or equal to 10). Other ranges are also possible.

Referring again to FIG. 1, in certain embodiments, a detector 150 ispositioned proximate intersection 130 and adjacent fluidic channel 120(e.g., such that detector 150 is configured and arranged to detect aparticle entering fluidic channel 120 via intersection 130). In someembodiments, the detector is selected from the group consisting ofoptical detectors (e.g., fluorescence detectors, refractive indexdetectors, visible light and/or UV detectors, microscopes), masssensors, capacitive sensors, resistive pulse sensors, electrical currentsensors, MEMS pressure sensors, acoustic sensors, ultrasonic sensors,and thermal sensors. In some embodiments, the detector is a suspendedmicrochannel resonator. In certain embodiments, detector 150 isconfigured and arranged to detect a particle entering fluidic channel120 at or proximate to intersection 130 such that, upon entry of theparticle into fluidic channel 120, at least one property (e.g.,magnitude of applied pressure) of the one or more of the pressuresource(s) is changed.

Referring again to FIG. 2A, in certain embodiments, a detector 150 ispositioned proximate second fluidic channel 120 (e.g., such thatdetector 150 is configured and arranged to detect a particle exiting (orabout to exit) second fluidic channel 120 via exit 130). In someembodiments, the detector is selected from the group consisting ofoptical detectors (e.g., fluorescence detectors, refractive indexdetectors, visible light and/or UV detectors, microscopes), masssensors, capacitive sensors, resistive pulse sensors, electrical currentsensors, MEMS pressure sensors, acoustic sensors, ultrasonic sensors,and thermal sensors. In some embodiments, the detector is a suspendedmicrochannel resonator. In certain embodiments, detector 150 isconfigured and arranged to detect a particle exiting (or about to exit)second fluidic channel 120 at or proximate to exit 130 such that, uponexit of the particle into first fluidic channel 110, at least oneproperty (e.g., magnitude of applied pressure) of the one or more of thepressure source(s) is changed. For example, in some embodiments, themagnitude of pressure applied by pressure source 190 may be increasedupon detection of a particle near exit 130, such that the particle iscaptured by the flow of a fluid in first fluidic channel 1 and/or sothat only a single particle is captured. In certain embodiments, theapplied pressure after capturing a single particle is increased suchthat no additional particles exit the exit of the second fluidic channelfor a desired amount of time (e.g., greater than or equal to 10seconds).

In some embodiments, the detector is located less than or equal to 5 mm,less than or equal to 4 mm, less than or equal to 3 mm, less than orequal to 2 mm, less than or equal to 1 mm, less than or equal to 900microns, less than or equal to 800 microns, less than or equal to 700microns, less than or equal to 600 microns, less than or equal to 500microns, less than or equal to 400 microns, less than or equal to 300microns, or less than or equal to 200 microns upstream from the exit ofthe second fluidic channel. In certain embodiments, the detector islocated greater than or equal to 100 microns, greater than or equal to200 microns, greater than or equal to 300 microns, greater than or equalto 400 microns, greater than or equal to 500 microns, greater than orequal to 600 microns, greater than or equal to 700 microns, greater thanor equal to 800 microns, greater than or equal to 900 microns, greaterthan or equal to 1 mm, greater than or equal to 2 mm, greater than orequal to 3 mm, or greater than or equal to 4 mm upstream from the exitof the second fluidic channel. Combinations of the above-referencedranges are also possible (e.g., less than or equal to 5 mm and greaterthan or equal to 100 microns). Other ranges are also possible.

As described herein, in some embodiments, a fluid comprising a pluralityof particles may be introduced to the first fluidic channel. In someembodiments, a single particle from the plurality of particles entersthe second fluidic channel intersecting the first fluidic channel (i.e.active loading regime). For example, referring now to FIG. 3A (e.g.,illustrating an active loading regime), fluid 135 comprising pluralityof particles 140 may be introduced to, and flowed within, first fluidicchannel 110. In some embodiments, at least a portion of fluid 135 (e.g.,comprising plurality of particles 140) is flowed in a first direction,as indicated by arrow 162. For example, first pressure source 190 may beconfigured such that at least a portion of fluid 135 in first fluidicchannel 110 flows in the first direction, as indicated by arrow 162. Incertain embodiments, at least a portion of fluid 135 (e.g., comprisingplurality of particle 140) is flowed in a second direction, as indicatedby arrow 164. For example, second pressure source 195 may be configuredsuch that at least a portion of fluid 135 in first fluidic channel 110flows in the second direction, as indicated by arrow 164. The term‘active loading regime’ as used herein generally refers to the stepscorresponding to the introduction of one or more particles into thesecond fluidic channel from the first fluidic channel, as describedherein. The term ‘flushing regime’ as used herein generally refers tothe steps corresponding to the flow of a fluid comprising a plurality ofparticles in the first fluidic channel, wherein no particles enter thesecond fluidic channel from the first fluidic channel, as describedherein.

In some embodiments, fluid 135 is flowed in direction 162 and direction164 such that at least a portion of fluid 135 enters channel 120 atintersection 130. In certain embodiments, exemplary particle 142 (e.g.,a single particle from plurality of particles 140) enters channel 120.Detector 150 may detect the entry of particle 142 into channel 120 atintersection 130 (or at a location, detection position 134, proximate toand downstream of intersection 130). In some embodiments, the detectionlocation (e.g., detection position 134) may be less than or equal to 5mm, less than or equal to 4 mm, less than or equal to 3 mm, less than orequal to 2 mm, less than or equal to 1 mm, less than or equal to 900microns, less than or equal to 800 microns, less than or equal to 700microns, less than or equal to 600 microns, less than or equal to 500microns, less than or equal to 400 microns, less than or equal to 300microns, or less than or equal to 200 microns downstream from theintersection between the first fluidic channel and the second fluidicchannel. In certain embodiments, the detection location is greater thanor equal to 100 microns, greater than or equal to 200 microns, greaterthan or equal to 300 microns, greater than or equal to 400 microns,greater than or equal to 500 microns, greater than or equal to 600microns, greater than or equal to 700 microns, greater than or equal to800 microns, greater than or equal to 900 microns, greater than or equalto 1 mm, greater than or equal to 2 mm, greater than or equal to 3 mm,or greater than or equal to 4 mm downstream from the intersectionbetween the first fluidic channel and the second fluidic channel.Combinations of the above-referenced ranges are also possible (e.g.,less than or equal to 5 mm and greater than or equal to 100 microns).Other ranges are also possible.

In some embodiments, a single particle (e.g., a single biologicalentity) may be separated from a plurality of particles. In someembodiments, the density of the plurality of particles in the fluid isgreater than or equal to 100 particles per milliliter, greater than orequal to 250 particles per milliliter, greater than or equal to 500particles per milliliter, greater than or equal to 1,000 particles permilliliter, greater than or equal to 2,500 particles per milliliter, orgreater than or equal to 5,000 particles per milliliter of fluid.Advantageously, a single particle (e.g., a single biological entity) maybe separated from a plurality of particles (e.g., plurality ofbiological entities) having a relatively large density of particles,using the method and systems described herein. For example, in someembodiments, the density of particles in the fluid is greater than orequal to 10,000 particles per milliliter, greater than or equal to25,000 particles per milliliter, greater than or equal to 50,000particles per milliliter, greater than or equal to 100,000 particles permilliliter, greater than or equal to 150,000 particles per milliliter,greater than or equal to 200,000 particles per milliliter, greater thanor equal to 250,000 particles per milliliter, greater than or equal to300,000 particles per milliliter, greater than or equal to 350,000particles per milliliter, greater than or equal to 400,000 particles permilliliter, greater than or equal to 450,000 particles per milliliter,greater than or equal to 500,000 particles per milliliter, greater thanor equal to 550,000 particles per milliliter, greater than or equal to600,000 particles per milliliter, greater than or equal to 650,000particles per milliliter, greater than or equal to 700,000 particles permilliliter, greater than or equal to 750,000 particles per milliliter,greater than or equal to 800,000 particles per milliliter, greater thanor equal to 850,000 particles per milliliter, greater than or equal to900,000 particles per milliliter, greater than or equal to 950,000particles per milliliter of fluid. In certain embodiments, the densityof particles in the fluid is less than or equal to 1,000,000 particlesper milliliter, less than or equal to 950,000 particles per milliliter,less than or equal to 900,000 particles per milliliter, less than orequal to 850,000 particles per milliliter, less than or equal to 800,000particles per milliliter, less than or equal to 750,000 particles permilliliter, less than or equal to 700,000 particles per milliliter, lessthan or equal to 650,000 particles per milliliter, less than or equal to600,000 particles per milliliter, less than or equal to 550,000particles per milliliter, less than or equal to 500,000 particles permilliliter, less than or equal to 450,000 particles per milliliter, lessthan or equal to 400,000 particles per milliliter, less than or equal to350,000 particles per milliliter, less than or equal to 300,000particles per milliliter, less than or equal to 250,000 particles permilliliter, less than or equal to 200,000 particles per milliliter, lessthan or equal to 150,000 particles per milliliter, less than or equal to100,000 particles per milliliter, less than or equal to 50,000 particlesper milliliter, or less than or equal to 25,000 particles per milliliterof fluid. In some embodiments, the density of particles in the fluid isless than or equal to 7,500 particles per milliliter, less than or equalto 5,000 particles per milliliter, less than or equal to 2,500 particlesper milliliter, less than or equal to 1,000 particles per milliliter,less than or equal to 500 particles per milliliter, or less than orequal to 250 particles per milliliter. Combinations of theabove-referenced ranges are also possible (e.g., greater than or equalto 100 particles per milliliter and less than or equal to 10,000particles per milliliter, greater than or equal to 100 particles permilliliter and less than or equal to 1,000,000 particles per milliliter,greater than or equal to 10,000 particles per milliliter and less thanor equal to 1,000,000 particles per milliliter, greater than or equal to10,000 particles per milliliter and less than or equal to 750,000particles per milliliter, greater than or equal to 500,000 particles permilliliter and less than or equal to 750,000 particles per milliliter).Other ranges are also possible. In an exemplary embodiment, the fluidcomprises a plurality of biological entities such as cells, and thedensity of cells within the fluid is greater than or equal to 10,000particles per milliliter and less than or equal to 750,000 particles permilliliter. Advantageously, the systems and methods described herein mayenable the loading of separated particle(s) into a channel at aparticular frequency (e.g., less than or equal to 1 particle per 10seconds) and/or spacing (e.g., greater than or equal to 1 mm)irrespective of the density of particles in the fluid, as compared topassive loading of cells into channels. That is to say, in someembodiments, substantially the same method and/or system may be used toseparate particles at a particular frequency and/or spacing within achannel, without diluting (or concentrating) the particles in the fluidprior to loading the fluid into the system. In some embodiments, therelatively high density of particles in the fluid are in a disorderedarrangement.

In certain embodiments, detection of a particle (e.g., a biologicalentity) in the second fluidic channel results in a change in at leastone property (e.g., magnitude of applied pressure) of at least onepressure source. Referring again to FIG. 3A, in some embodiments, auser, upon detection of a particle (e.g., exemplary particle 142) insecond fluidic channel 120 (e.g., at detection position 134), changes atleast one property of at least one pressure source. In certainembodiments, the change in at least one property occurs automatically(e.g., without user intervention) upon detection of a particle in thesecond fluidic channel intersecting the first fluidic channel.

In some embodiments, the change in at least one property of the at leastone pressure source changes the direction of flow of at least a portionof the fluid within the first fluidic channel. As illustrated in FIG. 3B(e.g., illustrating a flushing regime), upon detection of exemplaryparticle 142 by detector 150, at least a portion of fluid 135 flows in athird direction as indicated by arrow 166 (and different than the seconddirection as indicated by arrow 164 in FIG. 3A). In some embodiments,the first direction (indicated by arrow 162) and the third direction(indicated by arrow 166) is the same. In certain embodiments, upondetection of a particle in the second fluidic channel, the flow of thefluid (e.g., fluid 135) in the first fluidic channel is configured suchthat no additional particles in the plurality of particles enter thesecond fluidic channel from the first fluidic channel (i.e. the flushingregime). In some embodiments, the flushing regime comprises flowingfluid 135 in substantially the same direction.

Without wishing to be bound by theory, the pressure at the intersection(e.g., intersection 130) between the first fluidic channel (e.g., firstfluidic channel 110) and the second fluidic channel (e.g., secondfluidic channel 120), and/or the pressure drop along the second fluidicchannel, does not substantially change between the active loading regimeand the flushing regime. For example, in some embodiments, the fluidicpressure drop along the second fluidic channel during the flushingregime is within less than or equal to 10%, less than or equal to 8%,less than or equal to 6%, less than or equal to 4%, less than or equalto 2%, less than or equal to 1%, less than or equal to 0.5%, less thanor equal to 0.1%, or less than or equal to 0.05% of the fluidic pressuredrop along the second fluidic channel during the active loading regime.In certain embodiments, the fluidic pressure drop along the secondfluidic channel during the flushing regime is within greater than orequal to 0.01%, greater than or equal to 0.05%, greater than or equal to0.1%, greater than or equal to 0.5%, greater than or equal to 1%,greater than or equal to 2%, greater than or equal to 4%, greater thanor equal to 6%, or greater than or equal to 8% of the fluidic pressuredrop along the second fluidic channel during the active loading regime.Combinations of the above-referenced ranges are also possible (e.g.,less than or equal to 10% and greater than or equal to 0.01%). Otherranges are also possible. The change in speed at the intersection may bemeasured by measuring the speed of the loaded particles in secondfluidic channel 120, downstream of intersection 130 by detector 150 (orby a secondary detector associated with the second fluidic channel),where the change in speed of the particle is equal to the differencebetween the speed of the particle at the intersection during the activeloading regime and the speed of the particle during the flushing regime,expressed as a percentage of the speed of the particle at theintersection during the active loading regime. Without wishing to bebound by theory, the change in speed of the particle in the secondfluidic channel is proportional to the change in pressure drop along thesecond fluidic channel, such that the percent change in pressure drop isequivalent to the percent change in speed of the particle in the secondfluidic channel. Referring again to FIGS. 3A-3B, in an exemplaryembodiment, the pressure applied by pressure source 190 and pressuresource 195 during the active loading regime may be equal (e.g., suchthat fluid 135 flows towards second fluidic channel 120). In anotherexemplary embodiment, the pressure applied by pressure source 190 may begreater than the pressure applied by pressure source 195 during theflushing regime (e.g., such that fluid 135 flows in the same directionin first fluidic channel 110). For example, the pressure drop along thesecond fluidic channel is substantially the same whether the pressureapplied by pressure source 190 and pressure source 195 are equal orwhether the pressure applied by pressure source 190 and pressure source195 are unequal (e.g., the pressure applied by pressure source 190 maybe greater than the pressure applied by pressure source 195).

Advantageously, in some embodiments, the flow rate of the fluid in thesecond fluidic channel does not substantially change upon the change inat least one property of the at least one pressure source (e.g., suchthat at least a portion of the fluid in the first fluidic channelchanges direction). In certain embodiments, the particle (or at leastthe first particle) is maintained (or flowed) within the second fluidicchannel (e.g., during the flushing regime). In certain embodiments, theflow rate of the fluid (comprising the particle) in the second fluidicchannel during the flushing regime is within less than or equal to 10%,less than or equal to 8%, less than or equal to 6%, less than or equalto 4%, less than or equal to 2%, less than or equal to 1%, less than orequal to 0.5%, less than or equal to 0.1%, or less than or equal to0.05% of the flow rate of the fluid in the second fluidic channel duringthe active loading regime. In certain embodiments, the flow rate of thefluid in the second fluidic channel during the flushing regime is withingreater than or equal to 0.01%, greater than or equal to 0.05%, greaterthan or equal to 0.1%, greater than or equal to 0.5%, greater than orequal to 1%, greater than or equal to 2%, greater than or equal to 4%,greater than or equal to 6%, or greater than or equal to 8% of the flowrate of the fluid in the second fluidic channel during the activeloading regime. Combinations of the above-referenced ranges are alsopossible (e.g., less than or equal to 10% and greater than or equal to0.01%). Other ranges are also possible.

In some embodiments, it may be desirable to flow at least a portion ofthe fluid in the second channel back into the first channel (e.g., toremove a piece of undesirable debris and/or a second particle e.g.,detected by the detector). In some such embodiments, at least a portionof the fluid in which the single particle is suspended may be flowed outof the second channel and into the first channel while maintaining thesingle particle in the second channel. Those of ordinary skill in theart would understand, based upon the teachings of this specification,how to flow at least a portion of the fluid from the second channel intothe first channel. For example, a pressure source downstream of thesecond channel may apply a pressure to the second channel, such that atleast a portion of the fluid enters the first channel (while maintainingthe single particle in the second channel).

In some embodiments, it may be desirable to introduce two or moreparticles into the second channel. For example, in some embodiments, atleast a second particle (e.g., a second particle to be separated fromthe plurality of particles) may be flowed into the second fluidicchannel. For example, in certain embodiments, after an active loadingregime and a first particle enters the second fluidic channelintersecting the first fluidic channel (and is detected by thedetector), the system may enter a flushing regime (e.g., such that nomore particles enter the second fluidic channel). After a desired periodof time (and/or distance traveled along the second fluidic channel bythe first particle), the system may be switched back to the activeloading regime such that a second particle may enter the second fluidicchannel (e.g., at a particular frequency of entry and/or at a particularspacing from the first particle). Upon entry of the second particle intothe second fluidic channel (and detection by the detector), the systemmay return to a flushing regime, as described herein.

For example, as illustrated in FIG. 3C, once exemplary particle 142 hasflowed through second fluidic channel 120 for a desired length of time(or distance), the system can be switched back to an active loadingregime (e.g., such that at least a portion of fluid 135 is flowed in afirst direction as indicated by arrow 162 and at least a portion offluid 135 is flowed in a second direction, different than the firstdirection, as indicated by arrow 164). In some such embodiments, atleast one or more properties of the pressure source(s) (e.g., magnitudeof the applied pressure) may be changed (e.g., such that the pressureapplied by pressure source 190 and pressure source 195 is substantiallythe same). In certain embodiments, as illustrated in FIG. 3D, upondetection of a second exemplary particle (e.g., exemplary particle 144)by the detector in second fluidic channel 120, the system may beswitched to the flushing regime (e.g., such that no additional particlesenter second fluidic channel 120).

In some embodiments, the spacing between particles within the secondfluidic channel (e.g., distance 170 between exemplary particle 142 andexemplary particle 144 in second fluidic channel 120) may be controlled.For example, in some embodiments, exemplary particle 142 may be flowedin second fluidic channel 120 for a particular length of time (ordistance) under a flushing regime (e.g., such that no additionalparticles enter second fluidic channel 120). After a desired length oftime (or distance), the system may be switched back to an active loadingregime such that a second particle may enter second fluidic channel 120at a desired spacing and/or frequency. As described herein, the flowrate of the fluid (and/or particle) in the second fluidic channel duringthe flushing regime may not be significantly different than the flowrate of the fluid (and/or particle) in the second fluidic channel duringthe active loading regime.

Referring again to FIG. 2B, in some embodiments, the spacing betweenparticles within the second fluidic channel (e.g., distance 145 betweenexemplary particle 140 and exemplary particle 142 in second fluidicchannel 120) may be controlled. For example, in some embodiments,exemplary particle 140 may be flowed in second fluidic channel 120 for aparticular length of time (or distance) such that exemplary particle 140exits the second fluidic channel at exit 130 into first fluidic channel110. After a desired length of time (or distance), the exemplaryparticle 142 exits the second fluidic channel at exit 130 into firstfluidic channel 110. In some such embodiments, each particle may becollected (e.g., via outlet 170) separately from one another. In certainembodiments, the article, system, and/or device comprises a thirdfluidic channel, such that the second fluidic channel intersects thefirst fluidic channel and the third fluidic channel (e.g., the secondfluidic channel intersects and in disposed between the first fluidicchannel and the third fluidic channel). In some embodiments, the threechannels have a H-shaped geometry. One of ordinary skill in the artwould understand based upon the teachings of this specification thatother geometries are also possible. In some embodiments, the thirdfluidic channel comprises one or more pressure sources in fluidiccommunication with the third fluidic channel. For example, asillustrated in FIG. 3E, system 102 comprises first fluidic channel 110,third fluidic channel 112, and second fluidic channel 120 intersectingfirst fluidic channel 110 at intersection 130 and intersecting thirdfluidic channel 112 at intersection 131. In some embodiments, at leastone pressure source (e.g., third pressure source 192, fourth pressuresource 197) is associated with and/or in fluidic communication withthird fluidic channel 112. In some embodiments, the system may bedesigned such that the pressure at the intersection (e.g., intersection130) between the first fluidic channel and the second fluidic channel,and/or the flow rate within the second fluidic channel, may becontrolled, independent of the flow rate of the fluid in the firstchannel. In certain embodiments, a pressure may be applied (e.g., by oneor more pressure sources associated with and/or in fluidic communicationwith) to the third fluidic channel (e.g., third fluidic channel 112)such that the flow rate of the fluid in the second fluidic channel iscontrolled (e.g., maintained substantially) constant, independent to theflow rate of the fluid in the first fluidic channel. In someembodiments, the flow rate of the fluid in the second fluidic channel issubstantially similar during the active loading regime and the flushingregime. In some embodiments, the pressure may be applied to the thirdfluidic channel such that the average spacing between particles iscontrolled.

Referring again to FIG. 3D, in some embodiments, the average spacing(e.g., distance 170) between particles (e.g., exemplary particle 142 andexemplary particle 144) within the second fluidic channel (e.g., secondfluidic channel 120) may be at least 1 mm. For example, in certainembodiments, particles within the second fluidic channel may be spacedat an average spacing of at least 20 microns, at least 50 microns, atleast 100 microns, at least 250 microns, at least 500 microns, at least750 microns, at least 1 mm, at least 1.5 mm, at least 2 mm, at least 2.5mm, at least 3 mm, at least 4 mm, at least 5 mm, at least 10 mm, atleast 15 mm, at least 20 mm, at least 25 mm, at least 30 mm, at least 35mm, at least 40 mm, at least 45 mm, at least 50 mm, at least 75 mm, atleast 100 mm, at least 250 mm, or at least 400 mm apart along thelongitudinal axis of the second fluidic channel. In some embodiments,particles within the second fluidic channel may be spaced at an averagespacing of less than or equal to 500 mm, less than or equal to 400 mm,less than or equal to 250 mm, less than or equal to 100 mm, less than orequal to 75 mm, less than or equal to 50 mm, less than or equal to 45mm, less than or equal to 40 mm, less than or equal to 35 mm, less thanor equal to 30 mm, less than or equal to 25 mm, less than or equal to 20mm, less than or equal to 15 mm, less than or equal to 10 mm, less thanor equal to 5 mm, less than or equal to 4 mm, less than or equal to 3mm, less than or equal to 2.5 mm, less than or equal to 2 mm, less thanor equal to 1.5 mm, less than or equal to 1 mm, less than or equal to750 microns, less than or equal to 500 microns, less than or equal to250 microns, less than or equal to 100 microns, or less than or equal to50 microns apart along the longitudinal axis of the second fluidicchannel.

Combinations of the above-referenced ranges are also possible (e.g., atleast 20 microns and less than or equal to 500 mm, at least 20 micronsand less than or equal to 5 mm, at least 1 mm and less than or equal to50 mm, at least 1 mm and less than or equal to 500 mm). Other ranges arealso possible.

Referring again to FIG. 2B, in some embodiments, the average spacing(e.g., distance 145) between particles (e.g., exemplary particle 140 andexemplary particle 142) within the second fluidic channel (e.g., secondfluidic channel 120) may be at least 1 mm. For example, in certainembodiments, particles within the second fluidic channel may be spacedat an average spacing of at least 20 microns, at least 50 microns, atleast 100 microns, at least 250 microns, at least 500 microns, at least750 microns, at least 1 mm, at least 1.5 mm, at least 2 mm, at least 2.5mm, at least 3 mm, at least 4 mm, at least 5 mm, at least 10 mm, atleast 15 mm, at least 20 mm, at least 25 mm, at least 30 mm, at least 35mm, at least 40 mm, at least 45 mm, at least 50 mm, at least 75 mm, atleast 100 mm, at least 250 mm, or at least 400 mm apart along thelongitudinal axis of the second fluidic channel. In some embodiments,particles within the second fluidic channel may be spaced at an averagespacing of less than or equal to 500 mm, less than or equal to 400 mm,less than or equal to 250 mm, less than or equal to 100 mm, less than orequal to 75 mm, less than or equal to 50 mm, less than or equal to 45mm, less than or equal to 40 mm, less than or equal to 35 mm, less thanor equal to 30 mm, less than or equal to 25 mm, less than or equal to 20mm, less than or equal to 15 mm, less than or equal to 10 mm, less thanor equal to 5 mm, less than or equal to 4 mm, less than or equal to 3mm, less than or equal to 2.5 mm, less than or equal to 2 mm, less thanor equal to 1.5 mm, less than or equal to 1 mm, less than or equal to750 microns, less than or equal to 500 microns, less than or equal to250 microns, less than or equal to 100 microns, or less than or equal to50 microns apart along the longitudinal axis of the second fluidicchannel. Combinations of the above-referenced ranges are also possible(e.g., at least 20 microns and less than or equal to 500 mm, at least 20microns and less than or equal to 5 mm, at least 1 mm and less than orequal to 50 mm, at least 1 mm and less than or equal to 500 mm). Otherranges are also possible.

In certain embodiments, individual particles (e.g., biological entities)flowed in the second fluidic channel may be separated such that at least90% (e.g., at least 95%, at least 98%, at least 99%) of the spacingsdiffer by no more than less than 10%, less than 8%, less than 6%, lessthan 4%, less than 2%, or less than 1% of the average spacing betweenparticles. In some embodiments, individual particles flowed in thesecond fluidic channel may be separated such that at least 90% (e.g., atleast 95%, at least 98%, at least 99%) of the spacings differ by no morethan greater than or equal to 0.1%, greater than or equal to 1%, greaterthan or equal to 2%, greater than or equal to 4%, greater than or equalto 6%, or greater than or equal to 8% of the average spacing betweenparticles. In an exemplary embodiment, at least 90% of the particles areseparated such that the spacing between particles differs by no morethan 10% of the average spacing between the particles. Combinations ofthe above-referenced ranges are also possible (e.g., less than 10% andgreater than or equal to 0.1%). Other ranges are also possible. In someembodiments, the average spacing and/or the difference in spacingbetween particles is determined by measuring the spacing between 5 (ormore) consecutively loaded particles within the second fluidic channel.

In some cases, 2 or more, 5 or more, 10 or more, 20 or more, 50 or more,100 or more, 200 or more, 500 or more, or 750 or more particles (e.g.,biological entities) may be present in the second fluidic channel(and/or suspended microchannel resonators associated with the secondfluidic channel) at a given time. In certain embodiments, less than orequal to 1000, less than or equal to 750, less than or equal to 500,less than or equal to 200, less than or equal to 100, less than or equalto 50, less than or equal to 20, less than or equal to 10, or less thanor equal to 5 particles may be present in the second fluidic channel(and/or suspended microchannel resonators associated with the secondfluidic channel) at a given time. Combinations of the above-referencedranges are also possible (e.g., 2 or more and less than or equal to1000, 100 or more and less than or equal to 1000). Other ranges are alsopossible.

As illustrated in FIG. 5, in some embodiments, article 200 comprises aplurality of fluidically isolated surfaces (e.g., exemplary fluidicallyisolated surface 210 and isolated surface 212). In certain embodiments,a single particle (e.g., biological entity) is associated with eachfluidically isolated surface. For example, exemplary particle 142 isassociated with fluidically isolated surface 210 and exemplary particle144 is associated with fluidically isolated surface 212. In someembodiments, the plurality of particles (e.g., a plurality of biologicalentities) are provided (e.g., suspended) in a fluid (e.g., a liquid). Ina particular set of embodiments, the fluid is a liquid. In someembodiments, the fluid comprises water, a reagent, a solvent, a buffer,a cell-growth medium, or combinations thereof. In certain embodiments,the particles are relatively soluble in the fluid. In some embodiments,the fluid does not comprise a colloid (e.g., such as an emulsion). Forexample, in some embodiments, the particle (e.g., biological entity) isnot disposed (and/or encapsulated) by a first fluid that is immisciblewith, and surrounded by, a second fluid different than the first fluid.However, one of ordinary skill in the art, based upon the teachings ofthis specification, would understand that the systems and methodsdescribed herein may be used for separating a single colloid into achannel from a plurality of colloids.

Those of ordinary skill in the art would understand, based upon theteaching of this specification, that the single particle associated witha fluidically isolated surface may be in fluidic communication with thefluidically isolated surface. That is to say, the single particle may besuspended and/or in contact with a fluid that is also in contact withthe surface. The term “associated with” as used herein means generallyheld in close proximity, for example, a single particle associated witha fluidically isolated surface may be adjacent the surface. As usedherein, when a particle is referred to as being “adjacent” a surface, itcan be directly adjacent to (e.g., in contact with) the surface, or oneor more intervening components (e.g., a liquid) also may be present. Aparticle that is “directly adjacent” a surface means that no interveningcomponent(s) is present.

In some embodiments, the plurality of fluidically isolated surfaces maybe physically connected. For example, as illustrated in FIG. 6, article202 comprises a plurality of fluidically isolated surfaces (e.g.,exemplary fluidically isolated surface 2140 of well 220 and exemplaryfluidically isolated surface 2162 of well 222). In some embodiments, asingle particle (e.g., biological entity) is associated with eachfluidically isolated surface. For example, exemplary particle 242 isassociated with fluidically isolated surface 214 and exemplary particle244 is associated with fluidically isolated surface 216. While twoparticles and two fluidically isolated surfaces are illustrated in FIG.5 and FIG. 6, two or more fluidically isolated surfaces and/or separatedparticles may be present. For example, in some embodiments, the articlecomprises greater than or equal to 2, greater than or equal to 4,greater than or equal to 6, greater than or equal to 12, greater than orequal to 24, greater than or equal to 48, greater than or equal to 96,greater than or equal to 384 fluidically isolated surfaces. In certainembodiments, the article comprises less than or equal to 1536, less thanor equal to 384, less than or equal to 96, less than or equal to 48,less than or equal to 24, less than or equal to 12, less than or equalto 6, or less than or equal to 4 fluidically isolated surfaces.Combinations of the above-referenced ranges are also possible (e.g.,greater than or equal to 2 and less than or equal to 1536). Other rangesare also possible. In some embodiments, a plurality of particles areassociated with the fluidically isolated surfaces such that eachfluidically isolated surfaces is associated with a single particle (i.e.isolated particles) from the plurality of particles. In certainembodiments, isolated particles are associated with at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 98%, or atleast 99% of the fluidically isolated surfaces.

In certain embodiments, the article, system, and/or device comprises amulti-well plate (e.g., a multi-well cell culture plate) such as a6-well, 12-well, 24-well, 48-well, 96-well, 384-well, or 1536 wellplate. In some embodiments, the article comprises an ANSI multi-well(e.g., microtiter) plate. For example, in some cases, the article maycomprise a 96-well, 384-well, or 1536-well plate designed according theANSI SLAS 4-2004 (R2012) standard. In some embodiments, the articlecomprises a plurality of conical tubes (e.g., greater than or equal to 2and less than or equal to 1536 conical tubes). In certain embodiments,the article comprises a plurality of dishes such as petri dishes (e.g.,greater than or equal to 2 and less than or equal to 1536 petri dishes).

The fluidically isolated surfaces may comprise any suitable material.Non-limiting examples of suitable materials include polyethylene,polystyrene, polypropylene, cyclic olefin copolymers, vinyl (e.g.,polyvinyl chloride), and combinations thereof.

In some embodiments, the fluidically isolated surface is a cell-culturesurface (e.g., the surface(s) may be treated such that cells may adhereand/or grow on the surface). Cell-culture surfaces are known in the artand those of ordinary skill in the art would understand, based upon theteachings of this specification, how to select cell-culture surfaces foruse with the articles and methods described herein. For example, in someembodiments, at least a portion of the fluidically isolated surfaces maybe coated for cell-culture (e.g., a coating comprising collagen,poly-D-lysine, poly-L-lysine, gelatin, fibronectin, laminin, orcombinations thereof).

In some embodiments, the article, system, and/or device comprisesgreater than or equal to 2, greater than or equal to 4, greater than orequal to 6, greater than or equal to 12, greater than or equal to 24,greater than or equal to 48, greater than or equal to 96, greater thanor equal to 384 particles (e.g., biological entities), each particleassociated with a fluidically isolated surface. In certain embodiments,the article comprises less than or equal to 1536, less than or equal to384, less than or equal to 96, less than or equal to 48, less than orequal to 24, less than or equal to 12, less than or equal to 6, or lessthan or equal to 4 particles (e.g., biological entities), each particleassociated with a fluidically isolated surface. Combinations of theabove-referenced ranges are also possible (e.g., greater than or equalto 2 and less than or equal to 1536). Other ranges are also possible.

The particles may be separated from a relatively concentrated source ofparticles. In certain embodiments, the particles may be separatedwithout subsequent and/or significant dilution of the source and/orwithout the application of relatively high shear forces being applied tothe particles. For example, a relatively high density plurality ofparticles (e.g., biological entities) may be introduced to a fluidicchannel and a single particle from the plurality of particles may beflowed into an intersecting fluidic channel and separated from theplurality of particles without diluting the plurality of particles. Insome such cases, multiple particles may be flowed into the intersectingfluidic channel from the plurality of particles such that each particlein the intersecting fluidic channel are spaced apart with a relativelyuniform and large average spacing (e.g., at least 1 mm apart).

In an exemplary embodiment, as illustrated in FIG. 7, system 300comprises article 305 comprising a plurality of fluidically isolatedsurfaces (e.g., exemplary fluidically isolated surface 320 of well 310and fluidically isolated surface 322 of well 312). In some embodiments,system 300 comprises a fluidic channel 340 comprising a plurality ofparticles (e.g., particle 332 and particle 334) such that the particlesare spaced apart with a relatively uniform and large average spacing(e.g., at least 1 mm apart). Advantageously, the relatively large anduniform average spacing of the plurality of particles in the channel mayenable the isolation of single particles in single wells (or singlefluidically isolated surfaces) from a plurality of particles. Asillustrated in FIG. 7, exemplary particle 332 and particle 334 may bespaced apart at an average spacing (as indicated by distance 360).Distance 360 (e.g., the average spacing between particles in thechannel) is generally determined using the geometric center of eachparticle.

In some embodiments, each particle may be flowed in a fluidic channeland introduced to each fluidically isolated surface. Referring again toFIG. 7, in an exemplary embodiment, channel 340 comprises outlet 350,and outlet 350 may be positioned proximate well 312 such that particle332 may be introduced to well 312 and associated with fluidicallyisolated surface 322. In some embodiments, a fluid (e.g., a liquid) maybe present in channel 340 such that at least a portion of the fluid isintroduced to the fluidically isolated surface. In some embodiments, astage (e.g., a motorized stage) may be associated with the article(e.g., article 305) such that the article is moved with respect to theoutlet of the fluidic channel. In some such embodiments, the stage maybe moved after the introduction of a particle to each fluidicallyisolated surface, such that a single cell is introduced onto eachfluidically isolated surface. In some embodiments, a detector associatedwith the fluidic channel may be used to determine when a cell isapproaching the outlet of the channel such that the (next) fluidicallyisolated surface may be positioned proximate the outlet (such that asingle particle may be introduced to the fluidically isolated surface).

In some embodiments, a liquid introduced to the fluidically isolatedsurface with the particle (e.g., biological entity) has a relatively lowvolume. For example, in some embodiments, the volume of liquidassociated with the particle and the fluidically isolated surface isless than or equal to 100 microliters, less than or equal to 90microliters, less than or equal to 80 microliters, less than or equal to70 microliters, less than or equal to 60 microliters, less than or equalto 50 microliters, less than or equal to 40 microliters, less than orequal to 30 microliters, less than or equal to 20 microliters, less thanor equal to 10 microliters, less than or equal to 9 microliters, lessthan or equal to 8 microliters, less than or equal to 7 microliters,less than or equal to 6 microliters, less than or equal to 5microliters, less than or equal to 4 microliters, less than or equal to3 microliters, less than or equal to 2 microliters, less than or equalto 1 microliter, less than or equal to 0.5 microliters, or less than orequal to 0.2 microliters. In certain embodiments, the volume of liquidassociated with the particle and the fluidically isolated surface isgreater than or equal to 0.1 microliters, greater than or equal to 0.2microliters, greater than or equal to 0.5 microliters, greater than orequal to 1 microliter, greater than or equal to 2 microliters, greaterthan or equal to 3 microliters, greater than or equal to 4 microliters,greater than or equal to 5 microliters, greater than or equal to 6microliters, greater than or equal to 7 microliters, greater than orequal to 8 microliters, greater than or equal to 9 microliters, greaterthan or equal to 10 microliters, greater than or equal to 20microliters, greater than or equal to 30 microliters, greater than orequal to 40 microliters, greater than or equal to 50 microliters,greater than or equal to 60 microliters, greater than or equal to 70microliters, greater than or equal to 80 microliters, or greater than orequal to 90 microliters. Combinations of the above-referenced ranges arealso possible (e.g., greater than or equal to 0.1 microliter and lessthan or equal to 100 microliters, greater than or equal to 0.1microliter and less than or equal to 10 microliters). Other ranges arealso possible. In some cases, the device may be configured such that thevolume in which a single particle is collected from the outlet isrelatively low. For example, in some embodiments, the volume of liquidassociated with the particle collected from the outlet of the firstfluidic channel is less than or equal to 100 microliters, less than orequal to 90 microliters, less than or equal to 80 microliters, less thanor equal to 70 microliters, less than or equal to 60 microliters, lessthan or equal to 50 microliters, less than or equal to 40 microliters,less than or equal to 30 microliters, less than or equal to 20microliters, less than or equal to 10 microliters, less than or equal to9 microliters, less than or equal to 8 microliters, less than or equalto 7 microliters, less than or equal to 6 microliters, less than orequal to 5 microliters, less than or equal to 4 microliters, less thanor equal to 3 microliters, less than or equal to 2 microliters, lessthan or equal to 1 microliter, less than or equal to 0.5 microliters, orless than or equal to 0.2 microliters. In certain embodiments, thevolume of liquid associated with the particle collected from the outletof the first fluidic channel is greater than or equal to 0.1microliters, greater than or equal to 0.2 microliters, greater than orequal to 0.5 microliters, greater than or equal to 1 microliter, greaterthan or equal to 2 microliters, greater than or equal to 3 microliters,greater than or equal to 4 microliters, greater than or equal to 5microliters, greater than or equal to 6 microliters, greater than orequal to 7 microliters, greater than or equal to 8 microliters, greaterthan or equal to 9 microliters, greater than or equal to 10 microliters,greater than or equal to 20 microliters, greater than or equal to 30microliters, greater than or equal to 40 microliters, greater than orequal to 50 microliters, greater than or equal to 60 microliters,greater than or equal to 70 microliters, greater than or equal to 80microliters, or greater than or equal to 90 microliters. Combinations ofthe above-referenced ranges are also possible (e.g., greater than orequal to 0.1 microliter and less than or equal to 100 microliters,greater than or equal to 0.1 microliter and less than or equal to 10microliters). Other ranges are also possible.

In some embodiments, each single particle and associated liquid (e.g.,having a volume of less than or equal to 100 microliters) may becollected without discarding any intermediary fluid. For example, afirst particle and associated liquid may be collected from the outletand a second particle and associated liquid may be collected from theoutlet, without discarding any fluid between steps (e.g., collecting).Advantageously, the methods and devices described herein may have arelatively high uniformity of volume(s) collected with each particle. Insome embodiments, the volume of liquid collected with each of two ormore particles is substantially the same. In some embodiments, thedifference in volume of a fluid associated with a first particle and asecond particle does not vary by greater than or equal to 10% (e.g.,greater than or equal to 5%, greater than or equal to 2%, greater thanor equal to 1%) of the volume of each collected fluid. Advantageously,particles may be collected in relatively low and/or uniform volumes in,for example, two or more separate vessels.

In some embodiments, a single particle and associated liquid (e.g.,having a volume of less than or equal to 10 microliters) may beintroduced to each fluidically isolated surface without discarding anyfluid. For example, a first particle and associated liquid may beintroduction to a first fluidically isolated surface and a secondparticle and associated liquid may be introduced to a second fluidicallyisolated surface, without discarding any fluid between steps (e.g.,introduction). Advantageously, the methods and devices described hereinmay have a relatively high uniformity of volume across fluidicallyisolated surfaces. In some embodiments, the volume of liquid in two ormore fluidically isolated surfaces (comprising a single particleassociated with each fluidically isolated surfaces) is substantially thesame. In some embodiments, the difference in volume of a fluidassociated with two or more fluidically isolated surfaces of the articledoes not vary by greater than or equal to 10% (e.g., greater than orequal to 5%, greater than or equal to 2%, greater than or equal to 1%)of the volume of each fluidically isolated surface.

In some embodiments, at least one pressure source is associated withand/or in fluidic communication with the fluidic channel. Each pressuresource may comprise any suitable means for providing pressure to a fluiddisposed within the fluidic channel. For example, in some embodiments,each pressure source may be a pump such as a syringe pump, a suctionpump, a vacuum pump, or any other suitable pressure source. In someembodiments, each pressure source may not be in direct fluidiccommunication with the first fluidic channel. That is to say, in certainembodiments, one or more intervening fluidic channel(s) or fluidicregion(s) (e.g., fluidic reservoirs) of the device may be presentbetween the pressure source and the first fluidic channel.

In some embodiments, the individual particles flow in the fluidicchannel at a particular average velocity along the longitudinal axis ofthe fluidic channel. In certain embodiments, the average velocity of theparticles along the longitudinal axis of the fluidic channel is greaterthan or equal to 0.05 mm/second, greater than or equal to 0.1 mm/second,greater than or equal to 0.25 mm/second, greater than or equal to 0.5mm/second, greater than or equal to 0.75 mm/second, greater than orequal to 1 mm/second, greater than or equal to 2 mm/second, greater thanor equal to 3 mm/second, greater than or equal to 4 mm/second, greaterthan or equal to 5 mm/second, greater than or equal to 6 mm/second,greater than or equal to 7 mm/second, greater than or equal to 8mm/second, greater than or equal to 9 mm/second, greater than or equalto 10 mm/second, greater than or equal to 20 mm/second, greater than orequal to 30 mm/second, greater than or equal to 40 mm/second, greaterthan or equal to 50 mm/second, greater than or equal to 60 mm/second,greater than or equal to 70 mm/second, greater than or equal to 80mm/second, or greater than or equal to 90 mm/second. In someembodiments, the average velocity of the particles along thelongitudinal axis of the fluidic channel is less than or equal to 100mm/second, less than or equal to 90 mm/second, less than or equal to 80mm/second, less than or equal to 70 mm/second, less than or equal to 60mm/second, less than or equal to 50 mm/second, less than or equal to 40mm/second, less than or equal to 30 mm/second, less than or equal to 20mm/second, less than or equal to 10 mm/second, less than or equal to 9mm/second, less than or equal to 8 mm/second, less than or equal to 7mm/second, less than or equal to 6 mm/second, less than or equal to 5mm/second, less than or equal to 4 mm/second, less than or equal to 3mm/second, less than or equal to 2 mm/second, less than or equal to 1mm/second, less than or equal to 0.75 mm/second, less than or equal to0.5 mm/second, or less than or equal to 0.25 mm/second. Combinations ofthe above-referenced ranges are also possible (e.g., greater than orequal to 0.05 mm/second and less than or equal to 100 mm/second). Otherranges are also possible.

In some embodiments, the individual particles flow in the second fluidicchannel at a particular average velocity along the longitudinal axis ofthe second fluidic channel. In certain embodiments, the average velocityof the particles along the longitudinal axis of the second fluidicchannel is greater than or equal to 0.05 mm/second, greater than orequal to 0.1 mm/second, greater than or equal to 0.25 mm/second, greaterthan or equal to 0.5 mm/second, greater than or equal to 0.75 mm/second,greater than or equal to 1 mm/second, greater than or equal to 2mm/second, greater than or equal to 3 mm/second, greater than or equalto 4 mm/second, greater than or equal to 5 mm/second, greater than orequal to 6 mm/second, greater than or equal to 7 mm/second, greater thanor equal to 8 mm/second, or greater than or equal to 9 mm/second. Insome embodiments, the average velocity of the particles along thelongitudinal axis of the second fluidic channel is less than or equal to10 mm/second, less than or equal to 9 mm/second, less than or equal to 8mm/second, less than or equal to 7 mm/second, less than or equal to 6mm/second, less than or equal to 5 mm/second, less than or equal to 4mm/second, less than or equal to 3 mm/second, less than or equal to 2mm/second, less than or equal to 1 mm/second, less than or equal to 0.75mm/second, less than or equal to 0.5 mm/second, or less than or equal to0.25 mm/second. Combinations of the above-referenced ranges are alsopossible (e.g., greater than or equal to 0.05 mm/second and less than orequal to 10 mm/second). Other ranges are also possible.

In certain embodiments, the article, system, and/or device may beconfigured such that each particle (e.g., biological entity) enters thesecond fluidic channel from the first fluidic channel at a frequency ofless than or equal to 1 particle per 10 seconds, less than or equal to 1particle per 15 seconds, less than or equal to 1 particle per 20seconds, less than or equal to 1 particle per 25 seconds, less than orequal to 1 particle per 30 seconds, less than or equal to 1 particle per40 seconds, less than or equal to 1 particle per 50 seconds, less thanor equal to 1 particle per 60 seconds, less than or equal to 1 particleper 70 seconds, less than or equal to 1 particle per 80 seconds, lessthan or equal to 1 particle per 90 seconds, less than or equal to 1particle per 100 seconds, or less than or equal to 1 particle per 110seconds. In some embodiments, each particle enters the second fluidicchannel from the first fluidic channel at a frequency of greater than orequal to 1 particle per 120 seconds, greater than or equal to 1 particleper 110 seconds, greater than or equal to 1 particle per 100 seconds,greater than or equal to 1 particle per 90 seconds, greater than orequal to 1 particle per 80 seconds, greater than or equal to 1 particleper 70 seconds, greater than or equal to 1 particle per 60 seconds,greater than or equal to 1 particle per 50 seconds, greater than orequal to 1 particle per 40 seconds, greater than or equal to 1 particleper 30 seconds, greater than or equal to 1 particle per 25 seconds,greater than or equal to 1 particle per 20 seconds, greater than orequal to 1 particle per 15 seconds, or greater than or equal to 1particle per 10 seconds. Combinations of the above-referenced ranges arealso possible (e.g., less than or equal to 1 particle per 10 seconds andgreater than or equal to 1 particle per 120 seconds). Other ranges arealso possible.

In certain embodiments, the device may be configured such that eachparticle (e.g., biological entity) exits the fluidic channel into acollection channel at a frequency of less than or equal to 1 particleper 10 seconds, less than or equal to 1 particle per 15 seconds, lessthan or equal to 1 particle per 20 seconds, less than or equal to 1particle per 25 seconds, less than or equal to 1 particle per 30seconds, less than or equal to 1 particle per 40 seconds, less than orequal to 1 particle per 50 seconds, less than or equal to 1 particle per60 seconds, less than or equal to 1 particle per 70 seconds, less thanor equal to 1 particle per 80 seconds, less than or equal to 1 particleper 90 seconds, less than or equal to 1 particle per 100 seconds, orless than or equal to 1 particle per 110 seconds. In some embodiments,each particle exits a fluidic channel into a collection fluidic channelat a frequency of greater than or equal to 1 particle per 120 seconds,greater than or equal to 1 particle per 110 seconds, greater than orequal to 1 particle per 100 seconds, greater than or equal to 1 particleper 90 seconds, greater than or equal to 1 particle per 80 seconds,greater than or equal to 1 particle per 70 seconds, greater than orequal to 1 particle per 60 seconds, greater than or equal to 1 particleper 50 seconds, greater than or equal to 1 particle per 40 seconds,greater than or equal to 1 particle per 30 seconds, greater than orequal to 1 particle per 25 seconds, greater than or equal to 1 particleper 20 seconds, greater than or equal to 1 particle per 15 seconds, orgreater than or equal to 1 particle per 10 seconds. Combinations of theabove-referenced ranges are also possible (e.g., less than or equal to 1particle per 10 seconds and greater than or equal to 1 particle per 120seconds).

In certain embodiments, the system may be configured such that eachparticle (e.g., biological entity) is introduced to each fluidicallyisolated surface at a particular frequency (e.g., the time between eachparticle being introduced to each fluidically isolated surface via asingle outlet may be greater than or equal to 1 particle per 10seconds). In some embodiments, each particle is introduced to eachfluidically isolated surface at a frequency of

less than or equal to 1 particle per 10 seconds, less than or equal to 1particle per 15 seconds, less than or equal to 1 particle per 20seconds, less than or equal to 1 particle per 25 seconds, less than orequal to 1 particle per 30 seconds, less than or equal to 1 particle per40 seconds, less than or equal to 1 particle per 50 seconds, less thanor equal to 1 particle per 60 seconds, less than or equal to 1 particleper 70 seconds, less than or equal to 1 particle per 80 seconds, lessthan or equal to 1 particle per 90 seconds, less than or equal to 1particle per 100 seconds, or less than or equal to 1 particle per 110seconds. In some embodiments, each particle is introduced to eachfluidically isolated surface at a frequency of greater than or equal to1 particle per 120 seconds, greater than or equal to 1 particle per 110seconds, greater than or equal to 1 particle per 100 seconds, greaterthan or equal to 1 particle per 90 seconds, greater than or equal to 1particle per 80 seconds, greater than or equal to 1 particle per 70seconds, greater than or equal to 1 particle per 60 seconds, greaterthan or equal to 1 particle per 50 seconds, greater than or equal to 1particle per 40 seconds, greater than or equal to 1 particle per 30seconds, greater than or equal to 1 particle per 25 seconds, greaterthan or equal to 1 particle per 20 seconds, greater than or equal to 1particle per 15 seconds, or greater than or equal to 1 particle per 10seconds. Combinations of the above-referenced ranges are also possible(e.g., less than or equal to 1 particle per 10 seconds and greater thanor equal to 1 particle per 120 seconds). Other ranges are also possible.

In some embodiments, each single particle and associated liquid (e.g.,having a volume of less than or equal to 100 microliters) may becollected without discarding any intermediary fluid. For example, afirst particle and associated liquid may be collected from the outletand a second particle and associated liquid may be collected from theoutlet, without discarding any fluid between steps (e.g., collecting).Advantageously, the methods and devices described herein may have arelatively high uniformity of volume(s) collected with each particle. Insome embodiments, the volume of liquid collected with each of two ormore particles is substantially the same. In some embodiments, thedifference in volume of a fluid associated with a first particle and asecond particle does not vary by greater than or equal to 10% (e.g.,greater than or equal to 5%, greater than or equal to 2%, greater thanor equal to 1%) of the volume of each collected fluid. Advantageously,particles may be collected in relatively low and/or uniform volumes in,for example, two or more separate vessels.

In some embodiments, particles (e.g., biological entities) may be spacedwithin the second fluidic channel such that one or more properties ofthe particle (e.g., growth) may be monitored over relatively longperiods of time (e.g., greater than or equal to 10 minutes per particle)within the second fluidic channel. For example, in some embodiments, thesystems and methods described herein may be useful for providing cellsto a suspended microchannel resonator (or an array of suspendedmicrochannel resonators). For example, as illustrated in FIG. 4, system302 comprises first fluidic channel 110, second fluidic channel 120intersecting first fluidic channel 110 at intersection 130, detector150, and a suspended microchannel resonator 180 in fluidic communicationwith second fluidic channel 120.

In some embodiments, the articles and methods described herein may beuseful for collecting cells for which one or more properties had beenmeasured in a suspended microchannel resonator (or an array of suspendedmicrochannel resonators). For example, as illustrated in FIG. 2C, device102 comprises first fluidic channel 110, second fluidic channel 120orthogonal to first fluidic channel 110, exit 130 of second fluidic 120disposed within first fluidic channel 110, optional detector 150, and asuspended microchannel resonator 180 in fluidic communication withsecond fluidic channel 120.

In embodiments in which the system comprises one or more suspendedmicrochannel resonators, the suspended microchannel resonator may haveone or more characteristics described in commonly-owned U.S. Pat. No.7,387,889, entitled “Measurement of concentrations and bindingenergetics”, issued Jun. 17, 2008; commonly-owned U.S. Pat. No.7,838,284, entitled “Measurement of concentrations and bindingenergetics”, issued Nov. 23, 2010; commonly-owned U.S. Pat. No.9,134,294, entitled “Method And Apparatus For High Throughput DiagnosisOf Diseased Cells With Microchannel Devices”, issued Sep. 15, 2015;commonly-owned U.S. Pat. No. 9,134,295, entitled “Serial Arrays ofSuspended Microchannel Resonators”, issued Sep. 15, 2015; commonly-ownedU.S. Pat. No. 8.087,284, entitled “Method And Apparatus For MeasuringParticle Characteristics Through Mass Detection”, issued Jan. 3, 2012;commonly-owned U.S. Pat. No. 8,722,419, entitled “Flow cytometry MethodsAnd Immunodiagnostics With Mass Sensitive Readout”, issued May 13, 2014;each of which is incorporated herein by reference in its entirety forall purposes.

Fluids can be introduced (e.g., transport, flowed, displaced) into thesystem (or a fluidic channel therein (e.g., the first fluidic channel))using any suitable component, for example, a pump, syringe, pressurizedvessel, or any other source of pressure. Alternatively, fluids can bepulled into the fluidic channel by application of vacuum or reducedpressure on a downstream side of the channel or device. Vacuum may beprovided by any source capable of providing a lower pressure conditionthan exists upstream of the channel or device. Such sources may includevacuum pumps, venturis, syringes and evacuated containers. It should beunderstood, however, that in certain embodiments, methods describedherein can be performed with a changing pressure drop across a fluidicchannel by using capillary flow, the use of valves, or other externalcontrols that vary pressure and/or flow rate.

In some embodiments, introducing the fluid (e.g., comprising theplurality of particles) or at least a portion of the fluid comprisesapplying a pressure to the first fluidic channel such that at least aportion of a fluid enters the first fluidic channel. In certainembodiments, flowing the fluid comprises applying a pressure to thefirst fluidic channel such that at least a portion of a first fluid istransferred from (or to) the second fluidic channel intersecting thefirst fluidic channel. In certain embodiments, introducing the fluidcomprising the plurality of particles to the second fluidic channelcomprises applying a pressure to the second fluidic channel (or one ormore channel(s) in fluidic communication with the second fluidicchannel) such that at least a portion of the plurality of particlesenter the second fluidic channel. In some cases, the plurality ofparticles may be flowed such that one or more particles exit the secondfluidic channel at the exit of the second fluidic channel, and into thefirst fluidic channel, by applying a pressure to a fluid in the firstfluidic channel and/or a fluid in the second fluidic channel. In someembodiments, the pressure is a positive pressure. In certainembodiments, the pressure is a negative or reduced pressure.

In certain embodiments, flowing the fluid comprises applying a pressureto the first fluidic channel such that at least a portion of a fluid istransferred from (or to) the second fluidic channel orthogonal to (andat least partially disposed within) the first fluidic channel. In someembodiments, the pressure is a positive pressure. In certainembodiments, the pressure is a negative or reduced pressure.

One or more fluidic channels of the system may have any suitablecross-sectional shape (e.g., circular, oval, triangular, irregular,trapezoidal, square or rectangular, or the like). A fluidic channel mayalso have an aspect ratio (length to average cross sectional dimension)of at least 2:1, more typically at least 3:1, 5:1, or 10:1 or more. Afluid within the fluidic channel may partially or completely fill thefluidic channel.

In some embodiments, the one or more fluidic channels may have aparticular configuration. In certain embodiments, at least a portion ofone or more fluidic channels may be substantially linear in thedirection of fluid flow. In some embodiments, substantially all of oneor more fluidic channels is substantially linear in the direction offluid flow. In some embodiments, at least a portion of one or morefluidic channels may be curved, bent, serpentine, staggered, zig-zag,spiral, or combinations thereof. Advantageously, the use of a non-linearfluidic channels may permit the incorporation of one or more suspendedmicrochannel resonators into the system (e.g., in fluidic communicationwith at least the second fluidic channel).

The article(s), system(s), and device(s) or portions thereof (e.g., afluidic channel, a suspended microchannel resonator) described hereincan be fabricated of any suitable material. Non-limiting examples ofmaterials include polymers (e.g., polypropylene, polyethylene,polystyrene, poly(acrylonitrile, butadiene, styrene),poly(styrene-co-acrylate), poly(methyl methacrylate), polycarbonate,polyester, poly(dimethylsiloxane), PVC, PTFE, PET, or blends of two ormore such polymers), adhesives, and/or metals including nickel, copper,stainless steel, bulk metallic glass, or other metals or alloys, orceramics including glass, quartz, silica, alumina, zirconia, tungstencarbide, silicon carbide, or non-metallic materials such as graphite,silicon, or others.

In some embodiments, the fluid or system is maintained underphysiological conditions (e.g., for measuring cell growth). For example,in some embodiments, the fluid and/or the system is maintained at 37° C.and, optionally, pressurized with a 5% carbon dioxide gas mixture (e.g.,to maintain pH stability of the growth media).

EXAMPLES

The following examples are intended to illustrate certain embodimentsdescribed herein, including certain aspects of the present invention,but do not exemplify the full scope of the invention.

Example 1—Fluidic Operation of the Serial SMR Platform

The following example demonstrates the use of the system and methodsdescribed herein with an array of suspended microchannel resonators(SMRs) for growth rate and mass measurements of the cells. Asillustrated in FIG. 8A, an array of SMRs 470 is in fluidic communicationwith a first fluidic channel 450 and a second fluidic channel 460,intersecting first fluidic channel 450. A first pressure source 410 anda second pressure source 430 are located upstream of second channel 460,and each in fluidic communication with first fluidic channel 450.

The system has independent control of both upstream and downstreampressures applied to the first fluidic channel. This control enables,for example, the establishment of different volumetric flow rates alongthe first fluidic channel as compared to flow rate across the secondfluidic channel and mass sensor array (i.e. SMRs). In order to measuresingle-cell growth rates, a constant flow rate was maintained across thearray of mass sensors—i.e. P_(in)-P_(out) is maintained at a constantvalue for the entirety of a growth measurement experiment (asillustrated in corresponding FIG. 8B). FIG. 8B shows a resistor diagramfor the fluidic channels of the system. Based on the symmetry of thefluidic channel designs and the channels used to run fluid through thedevice all resistances (R1) in the first fluidic channel are equivalent(with the exception of the channel used to collect cells from the device(R2)). Seeing as this collection channel had a smaller inner diameter,it generally lead to a higher fluidic resistance (R2>R1). FIG. 8B alsoincludes the pressure values which determined the fluidic operation ofthe system including the upstream pressures (P1 and P2), downstreampressures (P3 and P4) and the pressures at the entrance and exit of theSMR mass sensor array (P_(in) and P_(out)).

For the majority of the experiment, the upstream and downstreampressures applied to the bypass fluidic channel on the cell-loading sideof the array were held constant in order to load cells in to the masssensor array (P1=P3). However, this fluidic balance lead to a very lowvolumetric flow rate—on the order of 1 μl per hour—in the first fluidicchannel. As such, for flushing the dead volume of the first fluidicchannel and loading a sample of cells in to the platform formeasurement, a significantly higher flow rate was generated along thecell-loading first fluidic channel (P1>>P3). During this flushingperiod, Pin was maintained at a constant value by increasing P1 anddecreasing P3 by the same value. In some cases, this ensured consistentflow speed across the mass sensor regardless of whether the cell-loadingfirst fluidic channel is in a state of cell (active) loading regimes orcell flushing regime as depicted in FIGS. 8A-8D. Depending on the typeof cell sample being measured, this flushing regime may also beimplemented periodically in order to deliver a fresh plug of cells formeasurement or clear any debris that may aggregate in the first fluidicchannel.

FIG. 8C shows a COMSOL fluidic model of the active loading regime for afirst fluidic channel and the second fluidic channel intersecting thefirst fluidic channel. A first pressure may be applied upstream of thefirst fluidic channel 450 at 410 (e.g., via a first pressure source) anda second pressure may be applied downstream of the first fluidic channel450 at 430 (e.g., via a second pressure source) such that the fluidenters second fluidic channel 460. FIG. 8D shows the flushing regime ofthe same system. The flow rates in the active loading regime and theflushing regimes within the second channel are substantially the same.

In some cases, it may be useful to ensure adequate spacing between cells(e.g., to control the collection of cells downstream). One approach tocontrolling the frequency of cell loading is to adjust the concentrationof cells that are loaded in to the array of mass sensors. Based onPoisson statistics, the volumetric flow rate and cell concentration perunit volume can be used to determine the average expected time betweencells entering the array (see ‘Passive Loading’ in FIG. 9). Althoughthis approach may be effective for limiting the number of co-collectionevents, it imposes inherent throughput limits on the platform. Forinstance, if the minimum time between cells required for collection is20 seconds, in order to reduce the co-collection frequency to less thanten percent, the cell concentration would have to be adjusted to yieldan average time between cells of roughly 60 seconds. This time may beincreased further when attempting to achieve a higher success rate forsingle-cell capture. As such, although the maximum throughput of thesystem as determined by the time required to flush cells is on the orderof 180 cells per hour, a dilution approach alone limits the throughputto just 60 cells per hour.

In order to address the limitations of concentration-based cell loading,an active loading regime was implemented for the serial SMR devices.This fluidic process uses active switching between the flushing andloading configurations presented in FIGS. 8A-8D. With real-time accessto the data generated by a detector (e.g., the first mass sensor in thearray), it is possible to determine when a cell has entered the arraybased on the corresponding shift in resonant frequency of that sensor.This frequency shift may be used to trigger a switch from the cell(active) loading regime to the cell flushing regime. Although thevolumetric sampling from the cell solution is equivalent between thesetwo modes—based on the consistent flow maintained across the array ofmass sensors—the volumetric flow fraction directed along the firstfluidic channel while flushing is significantly greater than the flowdirected to the array. Therefore, during the flushing regime, themajority of the streamlines continue along the first fluidic channel(FIG. 8D). Because cells are of finite size and occupy multiplestreamlines, they are directed along the first fluidic channel and arenot drawn in to the second fluidic channel. Therefore, as soon as thesystem is switched to a flushing regime, cells will not be loaded in tothe array. Once a cell (active) loading event is triggered and flushingbegins, a set amount of time is waited (e.g., 20 seconds) beforeswitching back to a cell loading regime. In order to capture a cellquickly after switching back to loading, a significantly higherconcentration cell sample may be used than is typical of experimentsrelying on Poisson based loading. Using this approach, a continuousstream of cells entering the array with a fixed separation in time, maybe enabled.

FIG. 9 shows plots of resonance frequency versus time for a mass sensor(e.g., a suspended microchannel resonator) for cells loaded into theintersecting fluidic channel using alternating active loading regimesand flushing regimes (“active loading”, top) and passive loading(bottom). A shift in resonant frequency is measured each time asingle-cell traverses the first mass sensor, each vertical spikeindicating a single-cell measurement. In the case of passive loading(top), single-cells enter the mass sensor array in aconcentration-dependent manner following a Poisson distribution, leadingto variability in the time spacing between sequential cell events. Foractive loading (bottom), the resonant frequency shift associated with asingle-cell entering the array is used to trigger a switch from theactive loading to flushing fluidic regimes (see FIGS. 8A-8D) until adesired time has elapsed and the system reverts back to a cell (active)loading regime. Such active switching allows for a substantially equallyspaced stream of cells may enter the array of mass sensors, as seen inFIG. 9.

Example 2—Isolation of Glioblastoma Cells on Fluidically IsolatedSurfaces

Glioblastoma BT145 cells at a concentration of 100,000 cells per mL weresorted into a 96-well plate, with a single glioblastoma cell in eachwell. Sphere forming assays were subsequently performed. FIGS. 10A-10Bare representative cell images from a first well including a firstfluidically isolated surface and a single cell associated with thesurface (FIG. 10A) and a second well including a second fluidicallyisolated surface and a single cell associated with the surface (FIG.10B). Images were taken immediately after cell release from the fluidicchannel of the device onto the fluidically isolated surface, and after 8days of cell culture. In this example, the single cell in FIG. 10Ademonstrated no growth, whereas the single cell in FIG. 10B demonstratedcell outgrowth.

Prophetic Example 1—Flow Focusing (Directing) for Rapid Cell Release(Collection)

As a single-cell exits the mass sensor array comprising a plurality ofsuspended microchannel resonators (e.g., via an exit of the secondfluidic channel) it enters the cell-collection channel (i.e. the firstfluidic channel). In a device in which the first and second fluidicchannel intersect, the first fluidic channel has a significantly greatervolumetric flow rate—a streamline distribution as seen in the fluidicmodel shown in FIG. 11 may occur. As such, the particle/cell wouldgenerally be driven very closely to the wall of the collection channelas it is being flushed from the device. Based on the flow profile withinthe collection channel, the particle/cell follows a fluidic path whichcauses it to move slower than the average velocity across the entirechannel. Therefore, rather than flushing the system for only long enoughto clear the dead volume of fluid—based on the volumetric flow rate—itinstead may need to be flushed for significantly longer in order toensure that the particle/cell is cleared from the system.

One way to achieve cell focusing at the center of the release channel iswith a varied fabrication approach, as illustrated in FIG. 12. Thecollection channel (i.e. the first fluidic channel) may be etched in aseparate layer—for instance in a top glass layer alone—such that theexit of the second fluidic channel can be positioned at or near thecenter of the particle/cell collection channel. This placement helpsparticles/cells exiting the mass sensor array to be centered in thecell-collection channel, thus placing the cells in the maximum flowprofile of the collection channel and/or helping to enable relativelyconsistent cell collection rates. In some cases, this approach mayreduce the minimum time required to flush a single particle/cell fromthe device and/or may increase the maximum achievable throughput of thecollection measurements.

While several embodiments of the present invention have been describedand illustrated herein, those of ordinary skill in the art will readilyenvision a variety of other means and/or structures for performing thefunctions and/or obtaining the results and/or one or more of theadvantages described herein, and each of such variations and/ormodifications is deemed to be within the scope of the present invention.More generally, those skilled in the art will readily appreciate thatall parameters, dimensions, materials, and configurations describedherein are meant to be exemplary and that the actual parameters,dimensions, materials, and/or configurations will depend upon thespecific application or applications for which the teachings of thepresent invention is/are used. Those skilled in the art will recognize,or be able to ascertain using no more than routine experimentation, manyequivalents to the specific embodiments of the invention describedherein. It is, therefore, to be understood that the foregoingembodiments are presented by way of example only and that, within thescope of the appended claims and equivalents thereto, the invention maybe practiced otherwise than as specifically described and claimed. Thepresent invention is directed to each individual feature, system,article, material, kit, and/or method described herein. In addition, anycombination of two or more such features, systems, articles, materials,kits, and/or methods, if such features, systems, articles, materials,kits, and/or methods are not mutually inconsistent, is included withinthe scope of the present invention.

The indefinite articles “a” and “an,” as used herein in thespecification and in the claims, unless clearly indicated to thecontrary, should be understood to mean “at least one.”

The phrase “and/or,” as used herein in the specification and in theclaims, should be understood to mean “either or both” of the elements soconjoined, i.e., elements that are conjunctively present in some casesand disjunctively present in other cases. Other elements may optionallybe present other than the elements specifically identified by the“and/or” clause, whether related or unrelated to those elementsspecifically identified unless clearly indicated to the contrary. Thus,as a non-limiting example, a reference to “A and/or B,” when used inconjunction with open-ended language such as “comprising” can refer, inone embodiment, to A without B (optionally including elements other thanB); in another embodiment, to B without A (optionally including elementsother than A); in yet another embodiment, to both A and B (optionallyincluding other elements); etc.

As used herein in the specification and in the claims, “or” should beunderstood to have the same meaning as “and/or” as defined above. Forexample, when separating items in a list, “or” or “and/or” shall beinterpreted as being inclusive, i.e., the inclusion of at least one, butalso including more than one, of a number or list of elements, and,optionally, additional unlisted items. Only terms clearly indicated tothe contrary, such as “only one of” or “exactly one of,” or, when usedin the claims, “consisting of,” will refer to the inclusion of exactlyone element of a number or list of elements. In general, the term “or”as used herein shall only be interpreted as indicating exclusivealternatives (i.e. “one or the other but not both”) when preceded byterms of exclusivity, such as “either,” “one of,” “only one of,” or“exactly one of.” “Consisting essentially of,” when used in the claims,shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase “atleast one,” in reference to a list of one or more elements, should beunderstood to mean at least one element selected from any one or more ofthe elements in the list of elements, but not necessarily including atleast one of each and every element specifically listed within the listof elements and not excluding any combinations of elements in the listof elements. This definition also allows that elements may optionally bepresent other than the elements specifically identified within the listof elements to which the phrase “at least one” refers, whether relatedor unrelated to those elements specifically identified. Thus, as anon-limiting example, “at least one of A and B” (or, equivalently, “atleast one of A or B,” or, equivalently “at least one of A and/or B”) canrefer, in one embodiment, to at least one, optionally including morethan one, A, with no B present (and optionally including elements otherthan B); in another embodiment, to at least one, optionally includingmore than one, B, with no A present (and optionally including elementsother than A); in yet another embodiment, to at least one, optionallyincluding more than one, A, and at least one, optionally including morethan one, B (and optionally including other elements); etc.

In the claims, as well as in the specification above, all transitionalphrases such as “comprising,” “including,” “carrying,” “having,”“containing,” “involving,” “holding,” and the like are to be understoodto be open-ended, i.e., to mean including but not limited to. Only thetransitional phrases “consisting of” and “consisting essentially of”shall be closed or semi-closed transitional phrases, respectively, asset forth in the United States Patent Office Manual of Patent ExaminingProcedures, Section 2111.03.

Any terms as used herein related to shape, orientation, alignment,and/or geometric relationship of or between, for example, one or morearticles, structures, forces, fields, flows, directions/trajectories,and/or subcomponents thereof and/or combinations thereof and/or anyother tangible or intangible elements not listed above amenable tocharacterization by such terms, unless otherwise defined or indicated,shall be understood to not require absolute conformance to amathematical definition of such term, but, rather, shall be understoodto indicate conformance to the mathematical definition of such term tothe extent possible for the subject matter so characterized as would beunderstood by one skilled in the art most closely related to suchsubject matter. Examples of such terms related to shape, orientation,and/or geometric relationship include, but are not limited to termsdescriptive of: shape—such as, round, square, circular/circle,rectangular/rectangle, triangular/triangle, cylindrical/cylinder,elliptical/ellipse, (n)polygonal/(n)polygon, etc.; angularorientation—such as perpendicular, orthogonal, parallel, vertical,horizontal, collinear, etc.; contour and/or trajectory—such as,plane/planar, coplanar, hemispherical, semi-hemispherical, line/linear,hyperbolic, parabolic, flat, curved, straight, arcuate, sinusoidal,tangent/tangential, etc.; direction—such as, north, south, east, west,etc.; surface and/or bulk material properties and/or spatial/temporalresolution and/or distribution—such as, smooth, reflective, transparent,clear, opaque, rigid, impermeable, uniform(ly), inert, non-wettable,insoluble, steady, invariant, constant, homogeneous, etc.; as well asmany others that would be apparent to those skilled in the relevantarts. As one example, a fabricated article that would described hereinas being “square” would not require such article to have faces or sidesthat are perfectly planar or linear and that intersect at angles ofexactly 90 degrees (indeed, such an article can only exist as amathematical abstraction), but rather, the shape of such article shouldbe interpreted as approximating a “square,” as defined mathematically,to an extent typically achievable and achieved for the recitedfabrication technique as would be understood by those skilled in the artor as specifically described. As another example, two or more fabricatedarticles that would described herein as being “aligned” would notrequire such articles to have faces or sides that are perfectly aligned(indeed, such an article can only exist as a mathematical abstraction),but rather, the arrangement of such articles should be interpreted asapproximating “aligned,” as defined mathematically, to an extenttypically achievable and achieved for the recited fabrication techniqueas would be understood by those skilled in the art or as specificallydescribed.

1-5. (canceled)
 6. A system, comprising: a first fluidic channel; asecond fluidic channel intersecting and in fluidic communication withthe first fluidic channel; at least one pressure source associated withthe first fluidic channel; and a detector associated with the secondfluidic channel, wherein the system is configured such that, upondetection by the detector of the presence of a single particle in thesecond fluidic channel, at least one property of one or more of the atleast one pressure source is changed.
 7. A system as in any claim 6,wherein the plurality of particles are a plurality of biologicalentities.
 8. A system as in claim 7, wherein the plurality of biologicalentities comprise virions, bacteria, protein complexes, exosomes, cells,or fungi.
 9. A system as in claim 6, wherein the first fluidic channelhas an average cross-sectional dimension of greater than or equal to 5microns and less than or equal to 2 mm.
 10. A system as in claim 6,wherein the second fluidic channel has an average cross-sectionaldimension of greater than or equal to 50 microns and less than or equalto 2 mm.
 11. A system as in claim 6, wherein the of the averagecross-sectional dimension of the first fluidic channel to the averagecross-sectional dimension of the second fluidic channel is at least 1and less than or equal to
 10. 12. A system as in claim 6, wherein adensity of particles in the first fluidic channel is greater than orequal to 100 particles per milliliter and less than or equal to1,000,000 particles per milliliter.
 13. A system as in claim 6, whereina fluidic pressure at the intersection during a flushing regime iswithin less than or equal to 10% and greater than or equal to 0.01% ofthe fluidic pressure at the intersection during an active loadingregime.
 14. A system as in claim 6, wherein a flow rate of the fluid inthe second fluidic channel during a flushing regime is within less thanor equal to 10% and greater than or equal to 0.01% of the flow rate ofthe fluid in the second fluidic channel during the active loadingregime.
 15. A system as in claim 6, wherein particles within the secondfluidic channel may be spaced at an average spacing of at least 20microns and less than or equal to 500 mm apart along a longitudinal axisof the second fluidic channel.
 16. A system as in claim 6, whereinindividual particles flowed in the second fluidic channel may beseparated such that at least 90% of the spacings differ by no more thanless than 10% and greater than or equal to 0.1%) of the average spacingbetween the particles.
 17. A system as in claim 6, wherein an averagevelocity of the particles along the longitudinal axis of the secondfluidic channel is greater than or equal to 0.1 mm/second and less thanor equal to 10 mm/second.
 18. A system as in claim 6, wherein eachparticle enters the second fluidic channel from the first fluidicchannel at a frequency of less than or equal to 1 particle per 10seconds and greater than or equal to 1 particle per 120 seconds.
 19. Asystem as in claim 6, wherein the particles are suspended in a fluid.20. A system as in claim 6, wherein the second fluidic channel is influidic communication with at least one suspended microchannelresonator.
 21. (canceled)
 22. A system as in claim 6, wherein thedetector is selected from the group consisting of optical detectors,mass sensors, capacitive sensors, thermal sensors, resistive pulsesensors, electrical current sensors, MEMS-based pressure sensors,acoustic sensors, ultrasonic sensors and suspended microchannelresonators. 23-52. (canceled)